A full-coverage line-scan raman microscopy imaging system and imaging method thereof

Abstract

The present application relates to biological microscopic imaging technology, and particularly relates to a full-coverage line scanning Raman microscopic imaging system and an imaging method thereof. The system comprises an excitation channel and a Raman channel. The excitation channel comprises a laser, a first convex lens, a second convex lens, a Powell prism, a first cylindrical lens, a second cylindrical lens, a beam splitter, a scanning galvanometer, a scanning lens, a sleeve lens, an objective lens and a sample stage. The Raman channel comprises a long-pass filter, a third convex lens, a slit, a fourth convex lens, a grating, a fifth convex lens and a CCD camera. The Raman light signals emitted by the biological sample are collected and returned to the beam splitter. The long-pass filter filters out the excitation wavelength light signals. The slit removes stray light outside the focal plane. The grating disperses in space and irradiates the CCD camera to obtain the measured Raman signals of the biological sample. The present application can quickly and accurately image the biological sample while reducing the illumination damage to the biological sample, and can provide strong help for biological and medical research and application.

Description

TECHNICAL FIELD

[0001] The present application relates to biological microscopic imaging technology, and in particular to a full-coverage line scanning Raman microscopic imaging system and an imaging method thereof. BACKGROUND

[0002] Raman spectrum is a non-destructive and non-labeled biological sample analysis technology, which can obtain structural and composition information of a sample at a molecular level. Compared with other spectral technologies such as infrared and fluorescence, Raman spectrum has advantages such as not being interfered by water, not being easily quenched, and narrow spectral bandwidth. Therefore, Raman spectrum is very suitable for detection of dynamic activities of biological samples.

[0003] The time scale of dynamic activities of biological samples (such as living cells) is several minutes to several hours. In order to capture the details of dynamic changes, the time resolution of the imaging system needs to reach the order of seconds or minutes. However, the spontaneous Raman scattering signal is at most only a thousandth of the scattering signal, and the Raman spectral signal is very weak.

[0004] Traditional point scanning Raman imaging systems usually need several hours to collect a frame of image, so they cannot capture the spatiotemporal evolution information in biological samples in time. Compared with point scanning, line scanning can improve the Raman imaging rate, but conventional line-by-line scanning still cannot perform Raman imaging on rapid activities, and the single position is damaged by long-term laser irradiation during a single measurement. Therefore, it is crucial to improve the conventional line-by-line scanning Raman imaging rate, accurately image the biological sample, and reduce the irradiation damage to the biological sample. SUMMARY

[0005] To solve the above problems, the technical scheme adopted by the present application is as follows: an imaging operation method for a Raman microscopic imaging system.

[0006] The specific technical scheme of the present application is as follows:

[0007] A full-coverage line scanning Raman microscopic imaging system, comprising an excitation channel, a Raman channel and a sample stage 12;

[0008] The excitation channel comprises a laser 1, a first convex lens 2, a second convex lens 3, a Powell prism 4, a first cylindrical lens 5, a second cylindrical lens 6, a beam splitter 7, a scanning galvanometer 8, a scanning lens 9, a sleeve lens 10, an objective lens 11 and a sample stage 12, and the sample stage 12 is used to place a biological sample for observation;

[0009] The point beam emitted by the laser 1 is changed in spot size by the first convex lens 2 and the second convex lens 3, and the spot is formed into a line beam by the Powell prism 4 and the first cylindrical lens 5 and the second cylindrical lens 6, and the line beam is coupled into the objective lens 11 through the scanning lens 9 and the sleeve lens 10, and is focused on the biological sample surface, and the line position selection of the line beam on the biological sample surface is realized by rotating the scanning galvanometer 8;

[0010] The Raman channel comprises a long-pass filter 13, a third convex lens 14, a slit 15, a fourth convex lens 16, a grating 17, a fifth convex lens 18 and a CCD camera 19.

[0011] The Raman light signal emitted by the biological sample is collected by the objective lens 11 and returned to the beam splitter 7, the excitation wavelength light signal is filtered out by the long-pass filter 13, the stray light outside the focal plane is removed by the slit 15, the light is dispersed in space by the grating 17, and is collected by the CCD camera 19, and the measurement Raman signal of the biological sample is obtained.

[0012] Further, the first convex lens 2 is placed along the direction of the light emitted by the laser 1, the second convex lens 3 is placed along the direction of the light emitted by the first convex lens 2, the Powell prism 4 is placed along the direction of the light emitted by the second convex lens 3, the first cylindrical lens 5 is placed along the direction of the light emitted by the Powell prism 4, the second cylindrical lens 6 is placed along the direction of the light emitted by the first cylindrical lens 5, the beam splitter 7 is placed along the direction of the light emitted by the second cylindrical lens 6, the scanning galvanometer 8 is placed along the direction of the reflected light of the beam splitter 7, the scanning lens 9 is placed along the direction of the light emitted by the scanning galvanometer 8, the sleeve lens 10 is placed along the direction of the light emitted by the scanning lens 9, and the objective lens 11 is placed along the direction of the light emitted by the sleeve lens 10; the sample stage 12 is located on the focal plane of the objective lens 11;

[0013] The long-pass filter 13 is placed along the direction of the light transmitted by the beam splitter 7, the third convex lens 14 is placed along the direction of the light emitted by the long-pass filter 13, the light emitted by the third convex lens 14 passes through the slit 15 to the fourth convex lens 16, the grating 17 is placed along the direction of the light emitted by the fourth convex lens 16, the fifth convex lens 18 is placed along the direction of the light emitted by the grating 17, and the CCD camera 19 is placed along the direction of the light emitted by the fifth convex lens 18. The imaging method of the full-coverage line scanning Raman microscopic imaging system also comprises the following steps: step (1): dividing a single image of a biological sample into a combination of N line positions, rotating the scanning galvanometer 8 to select the line positions, and selecting D line positions for the first Raman light signal integration detection within the single exposure time of the CCD camera 19, and the corresponding measurement matrix is A 1,1 , and the two-dimensional measurement Raman signal is H 1,1 ;

[0014] D line positions are randomly selected from the remaining ND line positions for a second Raman optical signal integration detection, with the corresponding measurement matrix being A. 1,2 The two-dimensional Raman signal obtained is H. 1,2 , ......

[0015] The measurement matrix corresponding to the k-th Raman optical signal integration detection is denoted as A. 1,k The measured two-dimensional Raman signal is denoted as H. 1,k Until the measurement of the N / Dth Raman optical signal integration detection is completed, the corresponding measurement matrix is ​​A. 1,K The two-dimensional Raman signal obtained is H. 1,K This allows N line positions to be detected once, thus completing a set of full-coverage compressed detection; where K = N / D, 1 ≤ k ≤ K;

[0016] Step (2): Repeating step (1) completes the m-th group of full-coverage compressed detection for a single image, and the corresponding measurement matrix is ​​denoted as A. m,k The two-dimensional Raman signal obtained is denoted as H. m,k A total of M sets of full-coverage compressed detections were performed, where the parameters satisfied the condition 1≤m≤M≤D; all the measurement matrices were spliced ​​together and denoted as A, and the corresponding two-dimensional measurement Raman signals were also spliced ​​together and denoted as H;

[0017] Step (3): The Raman signal obtained by compressed sensing can be described as a standard compressed sensing problem:

[0018] AS(λ)=H(λ) (1)

[0019] Where A is the compressed measurement matrix; S(λ) is the two-dimensional original Raman signal at Raman frequency shift λ; H(λ) is the two-dimensional measured Raman signal at Raman frequency shift λ;

[0020] Step (4): To compute S(λ) in Equation (1), solve the following minimization problem using the two-step iterative threshold shrinkage (TwIST) or the total variational enhanced Lagrange alternating direction (TVAL3) algorithm:

[0021]

[0022] In equation (2), the first term on the right side of the above equation is the fidelity term, and the second term on the right side is the regularization term based on total variation; τ is the adjustment parameter of the regularization term;

[0023] Step (5): Calculate the two-dimensional original Raman signals at all Raman frequency shifts λ. Combined together, they form a three-dimensional Raman data cube, which is the set of Raman signals at all locations on a single image of a biological sample;

[0024] The vertex component analysis method is used to obtain the Raman spectrum of various components in the biological sample and the abundance value of various components in space, i.e., a Raman image.

[0025] The beneficial technical effects of the present application are as follows:

[0026] The present application provides a full-coverage compressed line scanning Raman microscopic imaging system, which comprises an excitation channel and a Raman channel. The excitation channel excites the Raman light signal emitted by the biological sample, and the Raman channel collects the generated Raman light signal to obtain the measured Raman signal of the biological sample. The present application also provides a full-coverage compressed line scanning Raman microscopic imaging method. In single Raman signal detection, the line beam on the sample surface randomly selects several positions within the single exposure time of the CCD camera and stays for an equal time, and the Raman light signal integration detection is performed. In full-coverage compressed line scanning measurement, a single image is composed of N line positions, and D line positions are selected for single measurement. The residence time of the line beam at a single line position is shortened by D times compared with conventional line-by-line scanning detection. Not only the biological sample is rapidly imaged, but also the time consumption for collecting a frame is increased to the order of minutes or even seconds, which reduces the illumination damage to the biological sample and provides strong help for biological and medical research and application.

[0027] Meanwhile, in the full-coverage compressed detection of each group, it can be ensured that all positions in the plane are covered to avoid the loss of spatial information, and the Raman spectrum of various components in the biological sample and the Raman image of various components are obtained, so that the biological sample is accurately imaged. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a system diagram of the full-coverage compressed line scanning Raman microscopic imaging system of the present application;

[0029] Figure 2 It is a schematic diagram of the principle of conventional line-by-line scanning and compressed line scanning;

[0030] Figure 3 It is a schematic diagram of the measurement matrix of conventional line-by-line scanning;

[0031] Figure 4 It is a schematic diagram of the measurement matrix and line coverage statistics of conventional compressed line scanning;

[0032] Figure 5 It is a schematic diagram of the measurement matrix and line coverage statistics of full-coverage compressed line scanning;

[0033] Figure 6 It is a schematic diagram of the Raman image obtained by collecting the PS and PMMA mixed chemical microspheres by the three line scanning Raman imaging methods;

[0034] Figure 7are schematic diagrams of Raman spectra collected by three kinds of line-scan Raman imaging methods on PS and PMMA mixed chemical microspheres.

[0035] Wherein: laser 1, first convex lens 2, second convex lens 3, Powell prism 4, first cylindrical lens 5, second cylindrical lens 6, beam splitter 7, scanning galvanometer 8, scanning lens 9, sleeve lens 10, objective lens 11, sample stage 12, long-pass filter 13, third convex lens 14, slit 15, fourth convex lens 16, grating 17, fifth convex lens 18, CCD camera 19. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0037] Example 1

[0038] See Figure 1 A full-coverage line-scan Raman microscopic imaging system, comprising an excitation channel, a Raman channel and a sample stage 12; the excitation channel comprises a laser 1, a first convex lens 2, a second convex lens 3, a Powell prism 4, a first cylindrical lens 5, a second cylindrical lens 6, a beam splitter 7, a scanning galvanometer 8, a scanning lens 9, a sleeve lens 10, an objective lens 11 and the sample stage 12, and the sample stage 12 is used to place a biological sample for observation;

[0039] The point beam emitted by the laser 1 changes the spot size through the combination of the first convex lens 2 and the second convex lens 3, and the spot forms a line beam after passing through the combination of the Powell prism 4 and the first cylindrical lens 5 and the second cylindrical lens 6, and the line beam is coupled into the objective lens 11 through the scanning lens 9 and the sleeve lens 10, and is focused on the surface of the biological sample, and the line position selection of the line beam on the surface of the biological sample is realized by rotating the scanning galvanometer 8;

[0040] The Raman channel comprises a long-pass filter 13, a third convex lens 14, a slit 15, a fourth convex lens 16, a grating 17, a fifth convex lens 18 and a CCD camera 19;

[0041] The Raman light signal emitted by the biological sample is collected by the objective lens 11 and returned to the beam splitter 7, the excitation wavelength light signal is filtered out through the long-pass filter 13, the stray light outside the focal plane is removed through the slit 15, the stray light is dispersed in space through the grating 17, and the dispersed stray light is irradiated on the CCD camera 19 to be collected, so that the measurement Raman signal of the biological sample is obtained.

[0042] A first convex lens 2 is placed along the direction of the outgoing light rays of the laser 1, a second convex lens 3 is placed along the direction of the outgoing light rays of the first convex lens 2, a Powell prism 4 is placed along the direction of the outgoing light rays of the second convex lens 3, a first cylindrical lens 5 is placed along the direction of the outgoing light rays of the Powell prism 4, a second cylindrical lens 6 is placed along the direction of the outgoing light rays of the first cylindrical lens 5, a light splitting prism 7 is placed along the direction of the outgoing light rays of the second cylindrical lens 6, a scanning galvanometer 8 is placed along the direction of the reflected light rays of the light splitting prism 7, a scanning lens 9 is placed along the direction of the outgoing light rays of the scanning galvanometer 8, a sleeve lens 10 is placed along the direction of the outgoing light rays of the scanning lens 9, and an objective lens 11 is placed along the direction of the outgoing light rays of the sleeve lens 10; a sample stage 12 is located on the focal plane of the objective lens 11.

[0043] A long-pass filter 13 is placed along the direction of the transmitted light rays of the light splitting prism 7, a third convex lens 14 is placed along the direction of the outgoing light rays of the long-pass filter 13, the outgoing light rays of the third convex lens 14 pass through the slit 15 to a fourth convex lens 16, a grating 17 is placed along the direction of the outgoing light rays of the fourth convex lens 16, a fifth convex lens 18 is placed along the direction of the outgoing light rays of the grating 17, and a CCD camera 19 is placed along the direction of the outgoing light rays of the fifth convex lens 18. Figure 2 The schematic diagrams of the principles of the conventional line-by-line scanning and the compressed line scanning are shown in FIG. 2. The two modes are compared. In the single Raman signal detection, the line beam on the sample surface always stays at a certain position in the conventional line scanning mode, and the CCD camera 19 collects the Raman light signal generated at the position. In the compressed line scanning mode, the line beam on the sample surface randomly selects several positions by rotating the scanning galvanometer 8, and stays at each position for an equal time, and the CCD camera 19 integrates and collects the measured Raman light signals generated at the positions.

[0044] Embodiment 2

[0045] An imaging method of a full-coverage line scanning Raman microscopic imaging system, specifically comprising the following steps:

[0046] Step (1): dividing a single image of a biological sample into N line positions, rotating the scanning galvanometer 8 to select the line positions, and selecting D line positions in the single exposure time of the CCD camera 19 to perform the first Raman signal integration detection, and the corresponding measurement matrix is A 1,1 , and obtaining a two-dimensional measured Raman signal H 1,1 ;

[0047] Randomly selecting D line positions from the remaining N-D line positions to perform the second Raman signal integration detection, and the corresponding measurement matrix is A 1,2The two-dimensional Raman signal obtained is H. 1,2 , ......

[0048] The measurement matrix corresponding to the k-th Raman optical signal integration detection is denoted as A. 1,k The measured two-dimensional Raman signal is denoted as H. 1,k Until the measurement of the N / Dth Raman optical signal integration detection is completed, the corresponding measurement matrix is ​​A. 1,K The two-dimensional Raman signal obtained is H. 1,K This allows N line positions to be detected once, thus completing a set of full-coverage compressed detection; where K = N / D, 1 ≤ k ≤ K;

[0049] Step (2): Repeating step (1) completes the m-th group of full-coverage compressed detection for a single image, and the corresponding measurement matrix is ​​denoted as A. m,k The two-dimensional Raman signal obtained is denoted as H. m,k A total of M sets of full-coverage compressed detections were performed, where the parameters satisfied the condition 1≤m≤M≤D; all the measurement matrices were spliced ​​together and denoted as A, and the corresponding two-dimensional measurement Raman signals were also spliced ​​together and denoted as H;

[0050] Step (3): The Raman signal obtained by compressed sensing can be described as a standard compressed sensing problem:

[0051] AS(λ)=H(λ) (1)

[0052] Where A is the compressed measurement matrix; S(λ) is the two-dimensional original Raman signal at Raman frequency shift λ; H(λ) is the two-dimensional measured Raman signal at Raman frequency shift λ;

[0053] Step (4): To compute S(λ) in Equation (1), solve the following minimization problem using the two-step iterative threshold shrinkage (TwIST) or the total variational enhanced Lagrange alternating direction (TVAL3) algorithm:

[0054]

[0055] In equation (2), the first term on the right side of the above equation is the fidelity term, and the second term on the right side is the regularization term based on total variation; τ is the adjustment parameter of the regularization term;

[0056] Step (5): Calculate the two-dimensional original Raman signals at all Raman frequency shifts λ. Combined together, they form a three-dimensional Raman data cube, which is the set of Raman signals at all locations on a single image of a biological sample;

[0057] Using vertex component analysis, Raman spectra of various components in biological samples, as well as the spatial abundance values ​​of various components, i.e., Raman images, are obtained.

[0058] See Figure 3 In the conventional line-by-line scanning measurement, the line beam scans N = 100 line positions in a single direction one by one, and the Raman signal is detected one by one. After 100 detections, all line positions are covered, and full sampling of the image is achieved.

[0059] See Figure 4 In the conventional compressed line scanning measurement, a single image is composed of N = 100 line positions, and D = 5 line positions are randomly selected for a single measurement. The dwell time of the line beam at a single line position is 5 times shorter than that of the conventional line-by-line scanning detection, reducing the illumination damage to the biological sample. According to line coverage statistics, the number of coverages of each line position is from 0 to 10 times after 100 detections.

[0060] See Figure 5 In the full-coverage compressed line scanning measurement, a single image is composed of N = 100 line positions, and D = 5 line positions are selected for a single measurement. The dwell time of the line beam at a single line position is 5 times shorter than that of the conventional line-by-line scanning detection, reducing the illumination damage to the biological sample.

[0061] See Figure 6 The Raman images of the mixed 1 μm PS and 1 μm PMMA microspheres were obtained using the conventional line-by-line scanning, conventional compressed line scanning, and full-coverage compressed line scanning Raman imaging methods. A single image is composed of 100 line positions, and the exposure time of the CCD camera 19 is 1 second for a single measurement.

[0062] Taking the Raman image obtained by the conventional line-by-line scanning (sampling time 100 seconds) as the reference, the images obtained by the conventional compressed line scanning and the full-coverage compressed line scanning can maintain high fidelity when the compression ratio is in the range of 0 (sampling time 100 seconds) to 0.6 (sampling time 40 seconds). When the compression ratio reaches 0.8 (sampling time 20 seconds), the image obtained by the conventional compressed line scanning appears distorted, while the image obtained by the full-coverage compressed line scanning still maintains high fidelity. For example, a PMMA microsphere in the dotted box appears missing in the image obtained by the conventional compressed line scanning, but is still retained in the image obtained by the full-coverage compressed line scanning.

[0063] See Figure 7 The Raman spectra of the PS and PMMA mixed chemical microspheres were obtained using the conventional line-by-line scanning, conventional compressed line scanning, and full-coverage compressed line scanning Raman imaging methods. The vertex component analysis method was used to extract the Raman spectra of the conventional line scanning Raman image, the conventional compressed line scanning image with a compression ratio of 0.8 and 0.4, and the full-coverage compressed line scanning image in Figure 6 Taking the Raman spectrum obtained by the conventional line-by-line scanning as the reference, the Raman spectra obtained by the conventional compressed line scanning and the full-coverage compressed line scanning can maintain high fidelity.

[0064] It is to be understood that the above description is intended to be illustrative and not restrictive. Many other embodiments will be apparent to those of skill in the art upon reading and understanding the above description. The scope of the application should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. An imaging method for a full-coverage line-scan Raman microscopy imaging system, characterized in that: Specifically, the following steps are included: Step (1): Divide a single image of the biological sample into N line positions. Rotate the scanning galvanometer to select the line positions. During a single exposure time of the CCD camera, select D line positions for the first Raman light signal integration detection. The corresponding measurement matrix is ​​A. 1,1 The two-dimensional Raman signal obtained is H. 1,1 ; D line positions are randomly selected from the remaining ND line positions for a second Raman optical signal integration detection, with the corresponding measurement matrix being A. 1,2 The two-dimensional Raman signal obtained is H. 1,2 , ...... The measurement matrix corresponding to the k-th Raman optical signal integration detection is denoted as A. 1,k The two-dimensional Raman signal obtained is denoted as H. 1,k Until the measurement of the N / Dth Raman optical signal integration detection is completed, the corresponding measurement matrix is ​​A. 1,K The two-dimensional Raman signal obtained is H. 1,K , This ensures that N line positions are detected once, thus completing a set of full-coverage compressed detection; where K=N / D, 1≤k≤K; Step (2): Repeating step (1) completes the m-th group of full-coverage compressed detection for a single image, and the corresponding measurement matrix is ​​denoted as A. m,k The two-dimensional Raman signal obtained is denoted as H. m,k A total of M sets of full-coverage compressed detections were performed, where the parameters satisfied the condition 1≤m≤M≤D; all the measurement matrices were spliced ​​together and denoted as A, and the corresponding two-dimensional measurement Raman signals were also spliced ​​together and denoted as H; Step (3): The Raman signal obtained by compressed sensing can be described as a standard compressed sensing problem: （1） Where A is the compressed measurement matrix; S(λ) is the two-dimensional original Raman signal at Raman frequency shift λ; H(λ) is the two-dimensional measured Raman signal at Raman frequency shift λ; Step (4): To compute S(λ) in Equation (1), solve the following minimization problem using the two-step iterative threshold shrinkage (TwIST) or the total variational enhanced Lagrange alternating direction (TVAL3) algorithm: （2） In equation (2), the first term on the right side of the above equation is the fidelity term, and the second term on the right side is the regularization term based on total variation; It is the adjustment parameter for the regularization term; Step (5): The two-dimensional original Raman signals Ŝ(λ) at all Raman frequency shifts λ are combined together to form a three-dimensional Raman data cube, which is the set of Raman signals at all positions on a single image of a biological sample. Using vertex component analysis, Raman spectra of various components in biological samples, as well as the spatial abundance values ​​of various components, i.e., Raman images, are obtained.

2. The full-coverage line-scan Raman microscopy imaging system according to claim 1, characterized in that: Including excitation channels and Raman channels; The excitation channel includes a laser, a first convex lens, a second convex lens, a Powell prism, a first cylindrical mirror, a second cylindrical mirror, a beam splitter, a scanning galvanometer, a scanning lens, a sleeve lens, an objective lens, and a sample stage. The sample stage is used to place biological samples for observation. The spot beam emitted by the laser changes the spot size through a combination of a first convex lens and a second convex lens. The spot then forms a line beam after passing through a Powell prism and a combination of a first cylindrical mirror and a second cylindrical mirror. The line beam is coupled into the objective lens through a scanning lens and a sleeve lens and focused on the surface of the biological sample. Rotating the scanning galvanometer allows for the selection of the line position of the line beam on the surface of the biological sample. The Raman channel includes a long-pass filter, a third convex lens, a slit, a fourth convex lens, a grating, a fifth convex lens, and a CCD camera; The Raman light signal emitted by the biological sample is collected by the objective lens and returned to the beam splitter. The excitation wavelength light signal is filtered out by a long-pass filter, stray light outside the focal plane is removed by a slit, and the light is dispersed in space by a grating and then collected by a CCD camera to obtain the measured Raman signal of the biological sample.

3. The full-coverage line-scan Raman microscopy imaging system according to claim 2, characterized in that: A first convex lens is placed along the direction of the laser's emitted light; a second convex lens is placed along the direction of the emitted light from the first convex lens; a Powell prism is placed along the direction of the emitted light from the second convex lens; a first cylindrical mirror is placed along the direction of the emitted light from the Powell prism; a second cylindrical mirror is placed along the direction of the emitted light from the first cylindrical mirror; a beam splitter is placed along the direction of the emitted light from the second cylindrical mirror; a scanning galvanometer is placed along the direction of the reflected light from the beam splitter; a scanning lens is placed along the direction of the emitted light from the scanning galvanometer; a sleeve lens is placed along the direction of the emitted light from the scanning lens; and an objective lens is placed along the direction of the emitted light from the sleeve lens. The sample stage is located on the focal plane of the objective lens. A long-pass filter is placed along the direction of the transmitted light from the beam splitter. A third convex lens is placed along the direction of the emitted light from the long-pass filter. The emitted light from the third convex lens passes through the slit to a fourth convex lens. A grating is placed along the direction of the emitted light from the fourth convex lens. A fifth convex lens is placed along the direction of the emitted light from the grating. A CCD camera is placed along the direction of the emitted light from the fifth convex lens.

Images