CN109557070B - Raman imaging system based on space coded light - Google Patents
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Abstract
The invention belongs to the technical field of determination of physical or chemical properties or components of biological or medical samples, such as identification of unknown components of biological samples or analysis of medical clinical biopsy samples, and discloses a Raman imaging system based on spatial coding light; the continuous wave laser outputs a uniform plane irradiation light source after passing through the light source module and transmits the uniform plane irradiation light source to the space encoder; adopting a coding mode of a spatial coder to ensure that light passing through each pixel of the spatial coder has different intensity modulation frequencies; collecting time series Raman scattering signals by using a signal collecting module, and transmitting the signals to a control and calculation module for storage and subsequent processing; establishing a mathematical model of the spatial coding by utilizing the physical process of the spatial coding; and establishing a target function based on a sparse regularization strategy, and recovering the Raman image of the sample by adopting a proper optimization method. The invention combines the advantages of imaging speed of wide-field illumination and the advantages of image quality of point scanning, and has high image quality and high imaging speed.
Description
Technical Field
The present invention is in the field of analytical techniques for determining physical or chemical properties or components of biological or medical samples, e.g. identification of unknown components of biological samples or medical clinical biopsy samples, and in particular relates to a raman imaging system based on spatially coded light.
Background
Currently, the current state of the art commonly used in the industry is such that: at present, three modes of point scanning, line scanning and wide field illumination are mainly adopted for realizing Raman imaging. Point scanning raman imaging is the earliest occurring raman imaging mode, wherein exciting light is focused on one point on the surface of a sample to excite a raman scattering signal, the irradiation position of the sample is controlled through a galvanometer or a translation stage, and then the raman signal of each pixel point is recorded. Two-dimensional and three-dimensional imaging of a target sample can be realized based on the technology at present; however, because the raman scattering signal is weak, the excitation light dwell time per spot is typically on the order of milliseconds to seconds. In addition, data needs to be read after each acquisition, which typically adds hundreds of milliseconds to the signal detector. Thus, conventional point-scan raman imaging is a time-consuming process that may take several hours to map a small area. The linear scanning imaging mode has the speed increased by 300-600 times compared with the point scanning Raman imaging mode, exciting light is focused on the surface of a sample in a linear mode by the technology, Raman scattering signals excited by the exciting light are collected by a CCD camera, but the problems of image field bending, photon scattering and the like exist, so that an imaging object is slightly in front of the opposite focus point of the camera, imaging blur of the imaging object on two sides of the camera is caused, and acquisition of high-quality images is not facilitated. The wide-field illumination mode of Raman imaging technology adopts exciting light to irradiate the whole sample field, and then a CCD camera is adopted to acquire a two-dimensional Raman image of the sample. From the angle of imaging speed and image quality, the point scanning Raman imaging technology excites Raman signals by scanning each pixel point of a sample to enable the Raman signals to have the best image quality, but the point scanning Raman imaging technology is a time-consuming process and causes the imaging speed to be slow; the wide-field illumination raman imaging technology has a high imaging speed, but the whole sample is irradiated by directly using excitation light, so that the detailed chemical components and compositions of each pixel point are not easy to capture, and the image quality is slightly low.
In summary, the problems of the prior art are as follows: the traditional Raman imaging method has the defects of low imaging speed, image field bending, photon scattering and low image quality.
The difficulty and significance for solving the technical problems are as follows:
difficulty: the problems of low imaging speed in a point scanning mode, low imaging quality in a wide-field illumination mode, and image field bending and photon scattering in a line scanning mode are solved.
The significance is as follows: the advantages of high image quality of a point scanning mode and high imaging speed of a wide-field illumination mode are combined through a spatial coding mode, the problems in the prior art are solved, physical or chemical components and distribution of a biological or medical sample can be imaged quickly and with high quality, and more accurate analysis of a target sample is facilitated.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a Raman imaging system based on spatial coding light.
The invention is realized in such a way that a raman imaging system based on spatially coded light comprises:
the light source module is used for providing a high-quality, low-noise and stable uniform plane irradiation light source;
the spatial coding module is used for carrying out different frequency modulation on the light intensity at different positions of the plane irradiation light source by adopting a spatial coder;
the sample control module is used for controlling the sample to be in the optimal imaging position in six degrees of freedom;
the signal collection module is used for collecting Raman scattering optical signals generated after the sample is excited;
and the control and calculation module is used for controlling the light source module, the spatial coding module, the sample control module and the signal collection module and recovering the image of the spatial distribution of the Raman signal of the sample by adopting an imaging method based on spatial coding light.
Further, the light source module comprises a continuous wave laser and a light beam quality optimization and uniform plane irradiation light source generation submodule;
the spatial coding module comprises a spatial light modulator; the device is used for carrying out frequency modulation on the light intensity of a uniform plane irradiation light source emitted by a light source module, and ensuring that the light beam intensities of different irradiation positions of a sample have different modulation frequencies, so that Raman scattered light excited at each point of the sample has different modulation frequencies;
the sample control module comprises a sample bearing platform and a sample three-dimensional translation platform; the device is used for fixing a sample and adjusting the position of the sample to be at the optimal position of an imaging visual field;
the signal collection module comprises a notch filter, a large numerical aperture collection lens, a wavelength tunable filter and a high-sensitivity single-point optical signal detector; the Raman scattered light emitted after the sample is excited is firstly filtered by a notch filter to remove Rayleigh scattered light, then is collected by a large-numerical-aperture collecting lens, is received by a high-sensitivity single-point optical signal detector after the wavelength is selected by a wavelength tunable filter, and is transmitted to a control and calculation module for storage and subsequent processing after an optical signal is converted into an electric signal;
the control and calculation module comprises a computer control unit and a computer processing unit; the computer control unit comprises an image acquisition card and is connected with the high-sensitivity single-point optical signal detector and the space coding module; the control interface is connected with the light source module and the wavelength tunable filter;
the computer processing unit comprises an imaging method based on space coding light, is used for designing a coding mode of a space coder, and is used for carrying out image recovery on the Raman signals of the sample by utilizing the collected time series Raman scattering optical signals.
Another object of the present invention is to provide a spatially coded light based raman imaging method of a spatially coded light based raman imaging system, the spatially coded light based raman imaging method comprising the steps of:
the method comprises the following steps that firstly, a continuous wave laser outputs a uniform plane irradiation light source after passing through a light source module, and the uniform plane irradiation light source is transmitted to a space encoder;
designing a coding mode of the spatial coder to enable light passing through each pixel of the spatial coder to have different intensity modulation frequencies;
thirdly, collecting time series Raman scattering signals by using a signal collecting module, and transmitting the signals to a control and calculation module for storage and subsequent processing;
establishing a mathematical model of the spatial coding by using the physical process of the spatial coding, constructing a spatial coding matrix, and establishing a mathematical relation between the acquired time sequence Raman scattering signal and the two-dimensional image of the Raman signal of the sample to be recovered;
and fifthly, establishing a target function based on a sparse regularization strategy, and recovering the Raman image of the sample by adopting a proper optimization method.
Further, the second step specifically includes:
(1) determination of the maximum modulation frequency f from the single-point acquisition time tmax，
(2) Determining the time sequence acquisition point number N according to the pixel number N of the image to be restored,
(3) determining the lowest modulation frequency f from the number n of acquisition points of the time seriesmin，
(4) From the highest modulation frequency fmaxMinimum modulation frequency fminAnd the number N of the pixels of the image to be restored, and determining the modulation frequency resolution and the modulation frequency of each pixel.
Further, the fourth step specifically includes: establishing a mathematical model of spatial coding by using a physical process of the spatial coding, constructing a spatial coding matrix W, and establishing a mathematical relation between an acquired time sequence Raman scattering signal R and a two-dimensional image X of a sample Raman signal to be recovered:
R＝WX。
further, the construction of the mathematical relation between the spatial coding matrix W and the time series raman scattering signal R and the two-dimensional image X of the raman signal of the sample to be recovered comprises:
(1) marking an image to be restored as a two-dimensional matrix form, and converting the image to be restored into a column vector X according to a rule;
(2) marking the coding mode of the space coder at a fixed time point into a two-dimensional matrix form by using a weight mark, and converting the coding mode into a row vector W according to a rule;
(3) establishing a mathematical relation between the detection signal intensity of the high-sensitivity single-point optical signal detector, the coding mode of the space encoder and the image to be restored:
wherein i represents the ith acquisition time point;
(4) as time series data is acquired, the coding mode of the spatial coder changes, resulting in WiA change occurs; if the coding mode is changed n times, the following are:
(5) establishing a mathematical relation between the acquired time series Raman scattering signals and the two-dimensional image of the Raman signals of the sample to be recovered:
R＝WX；
in the formula, R is a single-point signal detector record value corresponding to the space coding mode coding n times; w is a spatial coding matrix, wherein each row represents a coding mode; x denotes the raman scattering image of the sample to be recovered.
Further, the step five objective function is:
minarg||R-WX||2+β||X||1；
where β is the regularization factor.
Another object of the present invention is to provide an application of the raman imaging system based on spatially coded light in battery nondestructive testing.
Another object of the present invention is to provide an application of the raman imaging system based on spatially coded light in nondestructive testing of biological materials.
The invention further aims to provide an application of the Raman imaging system based on the spatial coding light in graphene nondestructive testing.
In summary, the advantages and positive effects of the invention are: the Raman imaging system based on the space coding light has high image quality, can realize imaging in a multi-channel point scanning mode by adopting the space coder to modulate the intensity of different frequencies at different space positions of the plane irradiation light source, and greatly improves the image quality in a wide-field illumination mode. The Raman imaging system based on the space coding light has high imaging speed, and the simultaneous parallel imaging of a multi-channel point scanning mode can be realized in a space illumination mode by adopting the space encoder to modulate the intensity of different frequencies at different space positions of a plane irradiation light source, so that the imaging speed of the point scanning mode is greatly improved.
The invention adopts a space encoder to carry out different frequency modulation modes on the light intensity of different positions of a plane irradiation light source; the wide-field illumination imaging mode is provided, which is comparable to the point scanning imaging mode, combines the advantages of the wide-field illumination imaging speed and the point scanning image quality, and has the advantages of high image quality and high imaging speed. The method is simple to operate, easy to master and wide in application prospect. There can be contrast in speed, the dot scanning mode requires scanning one dot by one dot, if an image of 512 × 512 pixels is to be formed, 262144 times of scanning is required; whereas the wide field approach requires one acquisition. By adopting the system of the invention and combining sparse sampling, at least half of the time can be saved in data acquisition.
Drawings
Fig. 1 is a schematic structural diagram of a raman imaging system based on spatially coded light according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a Raman imaging system based on spatially encoded light according to an embodiment of the present invention;
in the figure: 1. a light source module; 1-1, a continuous laser; 1-2, generating a submodule for optimizing the quality of light beams and irradiating a light source on a uniform plane; 2. a spatial light modulator; 3. a sample control module; 4. a signal collection module; 4-1, a notch filter; 4-2, a large numerical aperture collection lens; 4-3, wavelength tunable filter (AOTF); 4-4, high sensitivity single point optical signal detector (PMT); 5. a control and calculation module; 5-1, a computer control unit; 5-2, and a computer processing unit.
Fig. 3 is a flowchart of a raman imaging method based on spatially coded light according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems of low imaging speed, image field bending, photon scattering and low image quality of the traditional Raman imaging method; the invention adopts a space encoder to carry out different frequency modulation on the light intensity of different positions of a plane irradiation light source; the wide-field illumination imaging mode which is comparable to point scanning is provided, and the quality and the imaging speed of a reconstructed image are improved; the chemical composition, concentration and distribution of each pixel point of the sample can be obtained, the recovery and reconstruction of the sample image are solved by adopting an optimization method based on sparse regularization, the recovered image quality is high, the imaging speed is high, and comprehensive sample information can be obtained.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
As shown in fig. 1 and fig. 2, a raman imaging system based on spatially coded light according to an embodiment of the present invention includes:
the light source module 1 is used for providing a high-quality, low-noise and stable uniform plane irradiation light source; the light source module 1 comprises a continuous wave laser 1-1 and a light beam quality optimization and uniform plane irradiation light source generation submodule 1-2.
The spatial coding module 2 is used for performing different frequency modulation on the light intensity of the plane irradiation light source at different positions by adopting a spatial coder; the spatial encoding module 2 comprises a spatial light modulator; the device is used for carrying out frequency modulation on the light intensity of a uniform plane irradiation light source emitted by the light source module 1, and ensuring that the light beam intensities of different irradiation positions of a sample have different modulation frequencies, so that Raman scattered light excited at each point of the sample has different modulation frequencies;
the sample control module 3 is used for controlling the sample to be in the optimal imaging position in six degrees of freedom; the sample control module 3 comprises a sample bearing platform and a sample three-dimensional translation platform; the device is used for fixing the sample and adjusting the position of the sample to be in the optimal position of the imaging field of view.
The signal collection module 4 is used for collecting Raman scattering optical signals generated after the sample is excited; the signal collection module 4 comprises a notch filter 4-1, a large numerical aperture collection lens 4-2, a wavelength tunable filter (AOTF)4-3 and a high-sensitivity single-point optical signal detector (PMT) 4-4; the Raman scattered light emitted after the sample is excited firstly filters Rayleigh scattered light through a notch filter, then is collected through a large-numerical-aperture collecting lens, is received by a high-sensitivity single-point optical signal detector after the wavelength is selected through a wavelength tunable filter, converts an optical signal into an electric signal, and transmits the electric signal to a control and calculation module 5 for storage and subsequent processing;
and the control and calculation module 5 is used for controlling the light source module 1, the spatial coding module 2, the sample control module 3 and the signal collection module 4 and for performing image recovery on the spatial distribution of the Raman signal of the sample by adopting an imaging method based on spatial coding light.
The control and calculation module 5 comprises a computer control unit 5-1 and a computer processing unit 5-2; the computer control unit 5-1 comprises an image acquisition card, and is connected with the high-sensitivity single-point optical signal detector 4-4 and the space coding module; and the control interface is connected with the light source module 1 and the wavelength tunable filter 4-3. The computer processing unit 5-2 comprises a spatially coded light based imaging method for designing the coding mode of the spatial encoder for image recovery of the sample raman signal using the acquired time series raman scattered light signal.
As shown in fig. 3, the raman imaging method based on spatially encoded light provided in the embodiment of the present invention includes the following steps:
s301: the continuous wave laser outputs a uniform plane irradiation light source after passing through the light source module and transmits the uniform plane irradiation light source to the space encoder;
s302: designing the coding mode of the spatial coder, so that the light passing through each pixel element of the spatial coder has different intensity modulation frequencies, and the coding mode of the spatial coder is different along with the time lapse;
s303: collecting time series Raman scattering signals by using a signal collecting module, and transmitting the signals to a control and calculation module for storage and subsequent processing;
s304: establishing a mathematical model of spatial coding by using a physical process of the spatial coding, constructing a spatial coding matrix, and establishing a mathematical relation between the acquired time sequence Raman scattering signal and a two-dimensional image of the Raman signal of the sample to be recovered;
s305: and establishing a target function based on a sparse regularization strategy, and recovering the Raman image of the sample by adopting a proper optimization method.
The Raman imaging method based on the space coding light provided by the embodiment of the invention specifically comprises the following steps:
step one, outputting and transmitting a light source; the continuous wave laser outputs a uniform plane irradiation light source after passing through the light source module and transmits the uniform plane irradiation light source to the space encoder;
designing a coding mode; designing the coding mode of the spatial coder, so that the light passing through each pixel element of the spatial coder has different intensity modulation frequencies, and the coding mode of the spatial coder is different along with the time lapse; the modulation frequency of each pixel of the spatial encoder is determined by single-point acquisition time and the number of pixels of an image to be restored according to the following principle:
(1) determination of the maximum modulation frequency f from the single-point acquisition time tmaxI.e. by
(2) Determining the number of acquisition points N of the time series from the number N of pixels of the image to be restored, i.e.
(3) Determining the lowest modulation frequency f from the number n of acquisition points of the time seriesminI.e. by
(4) From the highest modulation frequency fmaxMinimum modulation frequency fminAnd the number N of the pixels of the image to be restored, and determining the modulation frequency resolution and the modulation frequency of each pixel.
Step three, collecting and storing signals; collecting time series Raman scattering signals by using a signal collecting module, and transmitting the signals to a control and calculation module for storage and subsequent processing;
step four, establishing a model; establishing a mathematical model of spatial coding by using a physical process of the spatial coding, constructing a spatial coding matrix W, and establishing a mathematical relation between an acquired time sequence Raman scattering signal R and a two-dimensional image X of a sample Raman signal to be recovered:
R＝WX；
the construction of a mathematical relation between the spatial coding matrix W and the time series Raman scattering signal R and the two-dimensional image X of the Raman signal of the sample to be recovered comprises the following steps:
(1) marking an image to be restored as a two-dimensional matrix form, and converting the image to be restored into a column vector X according to a rule;
(2) marking the coding mode of the space coder at a fixed time point into a two-dimensional matrix form by using a weight mark, and converting the coding mode into a row vector W according to a rule;
(3) establishing a mathematical relation between the detection signal intensity of the high-sensitivity single-point optical signal detector, the coding mode of the space encoder and the image to be restored:
wherein i represents the ith acquisition time point;
(4) as time series data is acquired, the encoding mode of the spatial encoder changes, resulting in WiA change occurs; if the coding mode is changed n times, the following are:
(5) establishing a mathematical relation between the acquired time series Raman scattering signals and the two-dimensional image of the Raman signals of the sample to be recovered:
R＝WX；
in the formula, R is a single-point signal detector record value corresponding to the space coding mode coding n times; w is a spatial coding matrix, wherein each row represents a coding mode; x denotes the raman scattering image of the sample to be recovered.
Step five, restoring the image; establishing a target function based on a sparse regularization strategy, and recovering the Raman image of the sample by adopting a proper optimization method, wherein the target function is as follows:
minarg||R-WX||2+β||X||1；
where β is the regularization factor.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A spatially encoded light based raman imaging system, comprising:
the light source module is used for providing a high-quality, low-noise and stable uniform plane irradiation light source;
the spatial coding module is used for carrying out different frequency modulation on the light intensity at different positions of the plane irradiation light source by adopting a spatial coder;
the sample control module is used for controlling the sample to be in the optimal imaging position in six degrees of freedom;
the signal collection module is used for collecting Raman scattering optical signals generated after the sample is excited;
the control and calculation module is used for controlling the light source module, the spatial coding module, the sample control module and the signal collection module and recovering the image of the spatial distribution of the Raman signal of the sample by adopting an imaging method based on spatial coding light;
the light source module comprises a continuous wave laser and a light beam quality optimization and uniform plane irradiation light source generation submodule;
the spatial coding module comprises a spatial light modulator; the device is used for carrying out frequency modulation on the light intensity of a uniform plane irradiation light source emitted by a light source module, and ensuring that the light beam intensities of different irradiation positions of a sample have different modulation frequencies, so that Raman scattered light excited at each point of the sample has different modulation frequencies;
the sample control module comprises a sample bearing platform and a sample three-dimensional translation platform; the device is used for fixing a sample and adjusting the position of the sample to be at the optimal position of an imaging visual field;
the signal collection module comprises a notch filter, a large numerical aperture collection lens, a wavelength tunable filter and a high-sensitivity single-point optical signal detector; the Raman scattered light emitted after the sample is excited is firstly filtered by a notch filter to remove Rayleigh scattered light, then is collected by a large-numerical-aperture collecting lens, is received by a high-sensitivity single-point optical signal detector after the wavelength is selected by a wavelength tunable filter, and is transmitted to a control and calculation module for storage and subsequent processing after an optical signal is converted into an electric signal;
the control and calculation module comprises a computer control unit and a computer processing unit; the computer control unit comprises an image acquisition card and is connected with the high-sensitivity single-point optical signal detector and the space coding module; the control interface is connected with the light source module and the wavelength tunable filter;
the computer processing unit comprises an imaging method based on space coding light, is used for designing a coding mode of a space coder, and is used for carrying out image recovery on the Raman signals of the sample by utilizing the collected time series Raman scattering optical signals.
2. A spatially coded light based raman imaging method of operating the spatially coded light based raman imaging system of claim 1, characterized in that the spatially coded light based raman imaging method comprises the steps of:
the method comprises the following steps that firstly, a continuous wave laser outputs a uniform plane irradiation light source after passing through a light source module, and the uniform plane irradiation light source is transmitted to a space encoder;
designing a coding mode of the spatial coder to enable light passing through each pixel of the spatial coder to have different intensity modulation frequencies;
thirdly, collecting time series Raman scattering signals by using a signal collecting module, and transmitting the signals to a control and calculation module for storage and subsequent processing;
establishing a mathematical model of the spatial coding by using the physical process of the spatial coding, constructing a spatial coding matrix, and establishing a mathematical relation between the acquired time sequence Raman scattering signal and the two-dimensional image of the Raman signal of the sample to be recovered;
and fifthly, establishing a target function based on a sparse regularization strategy, and recovering the Raman image of the sample by adopting a proper optimization method.
3. The spatially coded light-based raman imaging method according to claim 2, wherein the second step specifically comprises:
(1) determination of the maximum modulation frequency f from the single-point acquisition time tmax，
(2) Determining the time sequence acquisition point number N according to the pixel number N of the image to be restored,
(3) determining the lowest modulation frequency f from the number n of acquisition points of the time seriesmin，
(4) From the highest modulation frequency fmaxMinimum modulation frequency fminAnd the number N of the pixels of the image to be restored, and determining the modulation frequency resolution and the modulation frequency of each pixel.
4. The raman imaging method based on spatially coded light according to claim 2, wherein said fourth step specifically comprises: establishing a mathematical model of spatial coding by using a physical process of the spatial coding, constructing a spatial coding matrix W, and establishing a mathematical relation between an acquired time sequence Raman scattering signal R and a two-dimensional image X of a sample Raman signal to be recovered:
R＝WX。
5. the spatially coded light based raman imaging method of claim 4, wherein the construction of the mathematical relationship between the spatial coding matrix W and the time series raman scattering signal R and the two-dimensional image X of the raman signal of the sample to be recovered comprises:
(1) marking an image to be restored as a two-dimensional matrix form, and converting the image to be restored into a column vector X according to a rule;
(2) marking the coding mode of the space coder at a fixed time point into a two-dimensional matrix form by using a weight mark, and converting the coding mode into a row vector W according to a rule;
(3) establishing a mathematical relation between the detection signal intensity of the high-sensitivity single-point optical signal detector, the coding mode of the space encoder and the image to be restored:
wherein i represents the ith acquisition time point;
(4) as time series data is acquired, the coding mode of the spatial coder changes, resulting in WiA change occurs; if the coding mode is changed n times, the following are:
(5) establishing a mathematical relation between the acquired time series Raman scattering signals and the two-dimensional image of the Raman signals of the sample to be recovered:
R＝WX；
in the formula, R is a single-point signal detector record value corresponding to the space coding mode coding n times; w is a spatial coding matrix, wherein each row represents a coding mode; x denotes the raman scattering image of the sample to be recovered.
6. The spatially coded light-based raman imaging method according to claim 2, wherein said step five objective function is:
minarg||R-WX||2+β||X||1；
where β is the regularization factor.
7. Use of the spatially coded light-based raman imaging system of claim 1 in battery non-destructive testing.
8. Use of a spatially coded light based raman imaging system according to claim 1 in the non-destructive testing of biological materials.
9. Use of the spatially coded light-based raman imaging system of claim 1 in graphene non-destructive testing.
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| Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection-Enhanced Topical Staining with SERS-Coded Nanoparticles;Wang, Yu W.;《SMALL》;20161026;第12卷(第40期);第5612-5621页 * |
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