A spectral microscopy imaging system and method for measuring unsaturation distribution of oils and fats

Abstract

The present invention relates to the field of optical microscopy, and more particularly to a spectral microscopy system and method for measuring the unsaturation distribution of oils and fats. The spectral microscopy system comprises a femtosecond laser, a first optical path component, a second optical path component, a beam combining optical path component, a CARS signal optical path component, and an image generation module. By introducing a half-wave plate and a quarter-wave plate into the optical path, the two beams required to generate the CARS signal are modulated into circularly polarized light with opposite rotational directions. The CARS spectrum intensity is calibrated using the non-resonant signal of pure water as a reference, thereby achieving the effect of measuring the unsaturation distribution of oils and fats. This method has the advantages of high signal intensity, low background noise, fast detection speed, high resolution, and non-destructiveness. It not only accurately measures the unsaturation of oils and fats, but also enables two-dimensional or three-dimensional microscopic imaging for detailed analysis of the unsaturation distribution in the sample. Furthermore, it can significantly improve the accuracy and efficiency of oil and fat quality analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopic imaging, and in particular to a spectral microscopic imaging system and method for measuring the unsaturation distribution of oils and fats. Background Art

[0002] Coherent anti-Stokes Raman scattering (CARS) utilizes the coherent optical signal generated by the resonance of the laser and the vibrational frequency of the sample molecules to selectively enhance the Raman signal of specific vibrational modes (such as the stretching vibration of the C-H bond). This method retains the advantage of Raman spectroscopy, which does not require complex sample preparation, while also producing signals several orders of magnitude stronger. Therefore, it offers higher sensitivity and faster detection speeds than Raman spectroscopy. Furthermore, because the generation of CARS signals is based on nonlinear optical effects and occurs only within the laser focal spot, which has a high energy density, this technique can provide submicron spatial resolution at video rates, enabling two-dimensional or three-dimensional microscopic imaging.

[0003] For these reasons, CARS has been applied to label-free microscopic imaging of lipids in biological samples, such as observing the intracellular distribution of lipids or studying temporal changes in intracellular lipid metabolism. However, due to the inevitable non-resonant background (NRB) in the CARS signal detection process, the intensity and peak shape of the CARS spectrum may be distorted, thus affecting the accuracy of quantitative analysis. In addition, the spectral focusing (SF) method commonly used to achieve CARS microscopic imaging can significantly improve its spectral resolution and image quality and realize multispectral imaging. However, the degree of temporal overlap of laser pulses in the SF process also affects the accuracy of CARS spectral intensity, which means that existing CARS microscopic imaging technology has not yet been applied to quantitatively measure the unsaturation and distribution of lipids.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a spectral microscopy imaging system and method for measuring the unsaturation distribution of oils and fats, aiming to solve the problem that the spectral microscopy imaging system cannot be used to quantitatively measure the unsaturation and distribution of oils and fats.

[0006] The technical solutions of the present invention are as follows:

[0007] A spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, comprising:

[0008] A femtosecond laser, configured to emit a first light beam and a second light beam that are different from each other; the first light beam is a pump light or a Stokes light, and the second light beam is a Stokes light or a pump light;

[0009] The first optical path component includes a first power adjustment module, a first glass rod, a first reflector group, a first beam expander, and a half-wave plate arranged in sequence along the first optical path;

[0010] The second optical path assembly includes a second power adjustment module, a second glass rod, a second reflector group, a second beam expander, a third reflector group, a time delay module, and a fourth reflector group, which are sequentially arranged along the second optical path;

[0011] A beam combining optical path assembly, comprising a first dichroic mirror for combining a first light beam passing through the first optical path assembly and a second light beam passing through the second optical path assembly, and a quarter wave plate, a scanner, a scanning lens, a tube lens, and a microscope objective lens sequentially arranged along the beam combining optical path; the microscope objective lens is used to focus the combined light beam on a sample surface and collect a CARS signal generated by the sample;

[0012] A CARS signal optical path component includes a second dichroic mirror, a filter, a lens, and a photomultiplier tube sequentially arranged along the CARS signal optical path;

[0013] An image generation module is used to generate a CARS image using the information obtained by the photomultiplier tube.

[0014] The spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, wherein the first power adjustment module is used to adjust the power and polarization direction of the first light beam; the second power adjustment module is used to adjust the power and polarization direction of the second light beam.

[0015] In the spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, the first glass rod is used to chirp and disperse the first light beam passing through the first power adjustment module into picosecond pulse light; and the second glass rod is used to chirp and disperse the second light beam passing through the second power adjustment module into picosecond pulse light.

[0016] In the spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, the first power adjustment module and the second power adjustment module are both composed of a half-wave plate and a polarization beam splitter.

[0017] In the spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, the time delay module is composed of a precision displacement stage and a pair of reflective mirrors fixed on the precision displacement stage.

[0018] In the spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, the half-wave plate and the quarter-wave plate are respectively fixed on a flip mirror frame.

[0019] A method for measuring the unsaturation distribution of oils and fats based on a spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats comprises the following steps:

[0020] The pump light and Stokes light are modulated into circularly polarized lights with opposite rotation directions. After adjusting the acquisition parameters, the CARS image at a specific wavenumber of the oil sample to be tested or the CARS image sequence corresponding to different wavenumbers is acquired;

[0021] Remove the half-wave plate and quarter-wave plate in the spectral microscopy imaging system, collect the baseline CARS image of pure water at the corresponding wavenumber, and calculate the average value of the image intensity as the calibration baseline intensity value;

[0022] The intensity value of each pixel in the CARS image or the CARS image sequence is divided by the correction reference intensity value for correction, and normalized to obtain a corrected CARS image;

[0023] The spectral intensity and image of the corrected CARS image at the specific wavenumber are analyzed to obtain the unsaturation and distribution of the oil in the oil sample to be tested.

[0024] In the method for measuring the unsaturation distribution of oils and fats, the acquisition parameters include laser power, scanning field of view, magnification, and acquisition frame number.

[0025] Beneficial effects: The present invention provides a spectral microscopy imaging system and method for measuring the unsaturation distribution of oils and fats. The spectral microscopy imaging system includes: a femtosecond laser for emitting a first light beam and a second light beam that are different from each other, as well as a first optical path component, a second optical path component, a beam combining optical path component, a CARS signal optical path component and an image generation module; the first light beam is pump light or Stokes light, and the second light beam is Stokes light or pump light. The present invention introduces a half-wave plate and a quarter-wave plate into an optical path to modulate the two beams of light required to generate a CARS signal into circularly polarized light with opposite rotation directions, thereby achieving the effect of removing the NRB effect in the CARS signal. A spectral microscopy system equipped with the half-wave plate and the quarter-wave plate is then used to collect a CARS image of a sample containing oil at a specific Raman frequency shift or a CARS image sequence corresponding to different wavenumbers. The half-wave plate and the quarter-wave plate are then removed, and while keeping all other acquisition parameters unchanged, a CARS image of pure water is collected at a corresponding wavenumber using the reference sample, and the average signal intensity is calculated as reference data for spectral intensity correction. The intensity value of each pixel in the CARS image of the oil sample is then divided by the intensity value of the pure water signal at the corresponding wavenumber, thereby correcting the CARS spectral intensity distortion caused by the different degrees of overlap of laser pulses in the time domain during the SF process and obtaining accurate CARS spectral intensity. Finally, the spectral microscopy system is used to measure the unsaturation distribution of the oil by analyzing the corrected CARS image at the specific wavenumber. In addition, this method has the advantages of high signal intensity, low background noise, fast detection speed, high resolution, and non-destructiveness. It can not only accurately measure the unsaturation of oils and fats, but also realize two-dimensional or three-dimensional microscopic imaging and analyze the distribution of unsaturation in the sample in detail; at the same time, it can significantly improve the accuracy and efficiency of oil quality analysis, providing a reliable new tool for food and other related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic structural diagram of a spectral microscopy imaging system for measuring unsaturation distribution of oils and fats according to the present invention;

[0027] Figure 2 Schematic diagram of the circular polarization modulation principle in the CARS microscopic imaging system of the present invention;

[0028] Figure 3 Schematic diagram of the pulse time interval adjustment principle in the SF-CARS system;

[0029] Figure 4 This is a flow chart of a method for measuring unsaturation distribution of oils and fats based on a spectral microscopy imaging system according to the present invention;

[0030] Figure 5 CARS spectra of EPA ME obtained under different situations;

[0031] Figure 6 Schematic diagram of the effect of using CARS images to analyze the distribution of oil unsaturation;

[0032] Explanation of the accompanying drawings: femtosecond laser 10, first light beam 11, second light beam 12, first power adjustment module 21, half-wave plate 211, polarization beam splitter 212, first glass rod 22, first reflector group 23, first beam expander 24, half-wave plate 25, second power adjustment module 31, second glass rod 32, second reflector group 33, second beam expander 34, third reflector group 35, time delay module 36, fourth reflector group 37, first dichroic mirror 41, 1 / 4 wave plate 42, scanner 43, scanning lens 44, tube lens 45, microscope objective lens 46, second dichroic mirror 51, filter 52, lens 53, photomultiplier tube 54. DETAILED DESCRIPTION

[0033] The present invention provides a spectral microscopy imaging system and method for measuring the unsaturation distribution of oils and fats. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0034] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0035] Measuring the unsaturation content of fats and oils is a key step in food quality control and nutritional analysis. Currently, widely used traditional measurement methods include the Wijs iodine method and gas chromatography. The Wijs iodine method involves reacting an iodine reagent with unsaturated fatty acids to form diiodide, which is then titrated to determine the degree of unsaturation. While simple and low-cost, the reaction conditions are demanding and the method only provides the unsaturated fatty acid content, providing no other information. Gas chromatography, on the other hand, converts the sample into a gaseous state, separates it on a chromatographic column, and analyzes it using a detector. While this method offers excellent separation and high sensitivity, it also requires complex sample pretreatment and requires a long detection time.

[0036] In addition to these two traditional methods, Raman spectroscopy, which exploits the Raman scattering phenomenon by illuminating a sample with a laser and detecting the frequency changes in the scattered light, can also be used to measure the unsaturation of oils and fats. This method requires no complex sample pretreatment, allows for in-situ non-destructive testing, and exhibits high sensitivity. However, due to the weak spontaneous Raman scattering signal, high background noise, and limited spatial resolution, it typically only provides single-point information on oil unsaturation, making it unsuitable for analyzing the distribution of unsaturation. Furthermore, existing CARS microscopy techniques have not yet been applied to quantitatively measure the unsaturation and distribution of oils and fats.

[0037] like Figure 1 As shown, the present invention provides a spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, comprising:

[0038] A femtosecond laser 10 is configured to emit a first light beam 11 and a second light beam 12 that are different from each other; the first light beam 11 is a pump light or a Stokes light, and the second light beam 12 is a Stokes light or a pump light;

[0039] The first optical path component includes a first power adjustment module 21, a first glass rod 22, a first reflector group 23, a first beam expander 24, and a half-wave plate 25 arranged in sequence along the first optical path;

[0040] The second optical path assembly includes a second power adjustment module 31, a second glass rod 32, a second reflector group 33, a second beam expander 34, a third reflector group 35, a time delay module 36, and a fourth reflector group 37, which are arranged in sequence in the second optical path;

[0041] A beam combining optical path assembly includes a first dichroic mirror 41 for combining a first light beam passing through the first optical path assembly and a second light beam passing through the second optical path assembly, as well as a quarter-wave plate 42, a scanner 43, a scanning lens 44, a tube lens 45, and a microscope objective 46 sequentially arranged along the beam combining optical path; the microscope objective 46 is used to focus the combined light beam on a sample surface and collect the CARS signal generated by the sample;

[0042] The CARS signal optical path component includes a second dichroic mirror 51, a filter 52, a lens 53, and a photomultiplier tube 54 arranged in sequence along the CARS signal optical path;

[0043] An image generation module is used to generate a CARS image using the information obtained by the photomultiplier tube.

[0044] In this embodiment, by introducing a half-wave plate and a quarter-wave plate into the optical path, the two beams required to generate the CARS signal are modulated into circularly polarized light with opposite rotation directions, thereby achieving the effect of removing the NRB effect in the CARS signal; then, a spectral microscopy imaging system equipped with a half-wave plate and a quarter-wave plate is used to collect a CARS image of a sample containing oil at a specific Raman frequency shift (expressed in wavenumber) or a CARS image sequence corresponding to different wavenumbers; then, the half-wave plate and the quarter-wave plate are removed, and pure water is used as the reference sample while keeping all other acquisition parameters unchanged. CARS images at the corresponding wavenumbers are collected and the average signal intensity is calculated, serving as the baseline data for spectral intensity correction. Next, the intensity value of each pixel in the CARS image of the oil sample is divided by the pure water signal intensity value at the corresponding wavenumber. This corrects the CARS spectral intensity distortion caused by the varying degrees of temporal overlap of the laser pulses during the SF process, thereby obtaining accurate CARS spectral intensity. Finally, by analyzing the corrected CARS images at specific wavenumbers, the spectral microscopy imaging system (SF-CARS system) is used to measure the unsaturation distribution of oils and fats. This method offers the advantages of high signal intensity, low background noise, fast detection speed, high resolution, and non-destructiveness. It not only accurately measures the unsaturation of oils and fats, but also enables two-dimensional or three-dimensional microscopic imaging for detailed analysis of the unsaturation distribution in the sample. Furthermore, it can significantly improve the accuracy and efficiency of oil and fat quality analysis, providing a reliable new tool for food and other related industries.

[0045] Specifically, since the pump light and Stokes light in the commonly used SF-CARS system are usually linearly polarized lights with the same polarization direction, Figure 2 As shown (where Figure 2 In the example, light beams 1 and 2 are combined: one represents the pump light and the other represents the Stokes light. A half-wave plate is introduced into the optical path of one of the beams to rotate its fast axis so that the polarization direction of the beam changes by 90 degrees. A quarter-wave plate is then introduced into the optical path after the beam combination to rotate its fast axis so that the polarization direction of the two beams differs by ±45 degrees, thereby modulating the two beams into left-handed and right-handed circularly polarized light, respectively. Furthermore, in the spectral microscopy imaging system, the two laser beams required to generate the CARS signal are provided by the same femtosecond pulse laser. One beam has a central wavelength of 1040 nm and serves as the Stokes light, while the other has a central wavelength of 800 nm and serves as the pump light. The corresponding central Raman frequency shift of the two beams is approximately 2885 cm -1 , considering that the line width of the two beams is about 150cm -1 The system can detect Raman frequency shifts in the range of approximately 2735-3035 cm -1This wavenumber range is related to the various vibration modes of the C-H bond, and oils are rich in C-H bonds. Therefore, a significant CARS signal can be detected using this system. The central wavelength of the generated CARS signal is about 650nm and the bandwidth is about 40nm.

[0046] In some embodiments, the first power adjustment module 21 is configured to adjust the power and polarization direction of the first light beam; the second power adjustment module 22 is configured to adjust the power and polarization direction of the second light beam. The first light beam and the second light beam are respectively adjusted in power by the first power adjustment module and the second power adjustment module, and are adjusted to have linearly polarized light with the same polarization direction.

[0047] In some embodiments, the first glass rod is used to chirp and disperse the first light beam passing through the first power regulation module into picosecond pulses; the second glass rod is used to chirp and disperse the second light beam passing through the second power regulation module into picosecond pulses. Chirp dispersion into picosecond pulses via the glass rods achieves SF-CARS.

[0048] Specifically, if Figure 3 As shown, the SF-CARS of the present invention can significantly improve the spectral resolution and image quality of CARS detection by stretching femtosecond pulses into picosecond pulses and adjusting the time interval between the two pulses to match the vibration mode of the sample, and realize the acquisition of Raman scattering signals of different wavenumbers (i.e., spectral scanning).

[0049] In some embodiments, the first power adjustment module 21 and the second power adjustment module 31 are both composed of a half-wave plate 211 and a polarization beam splitter 212. The power adjustment module composed of a half-wave plate and a polarization beam splitter can adjust the power and polarization direction of light.

[0050] In some embodiments, the time delay module is composed of a precision translation stage and a pair of mirrors fixed to the precision translation stage, which is used to change the optical path of the optical path, thereby accurately adjusting the overlap of the pump light and Stokes light pulses in the time domain to generate frequency differences corresponding to different vibration modes (i.e., Raman frequency shifts). By continuously moving the precision translation stage, frequency sweep detection of the CARS spectrum can be achieved.

[0051] Specifically, the adjustment of the pulse time interval can be achieved by using a delay line or other optical devices. In this embodiment, the position of the moving platform and the reflector is changed to change the optical path of one of the optical paths, thereby changing the pulse arrival time. However, the degree of overlap of the two pulses under different time interval conditions is different, especially at the two ends of the spectral scanning range, such as Figure 3As shown in the two cases on the left and right sides of the figure, the CARS signal intensity generated by the two pulses is also affected. Specifically, when the Raman shift is Ω1 and Ω3, the two pulses have only a small degree of temporal overlap, resulting in a significantly weaker CARS signal than when the Raman shift is Ω2. This affects the shape of the CARS spectrum and, in turn, the subsequent quantitative analysis based on spectral intensity. Therefore, for measuring the unsaturation distribution of oils and fats, pure water, which contains no =CH bonds, is used as a reference sample. Its NRB signal is measured under linear polarization conditions to measure the degree of temporal overlap between the two pulses. Specifically, at the ends of the spectral scan range, the two pulses do not completely overlap, reducing the effective excitation energy and thus weakening the pure water NRB signal, which is proportional to this energy. Therefore, by measuring the distribution of calibration intensity values ​​at different wavenumbers (corresponding to different temporal overlap conditions), the CARS signal intensity obtained when measuring the sample under test is divided by this calibration intensity value and renormalized to correct for the effect of varying pulse overlap on the CARS spectral intensity.

[0052] It should be noted that Figure 3 In the figure, the ellipses in the upper and lower rows represent the pump light and Stokes light pulses after broadening, respectively; ω p 、ω s represent the center frequencies of the pump light and Stokes light pulses respectively; Ω1, Ω2, and Ω3 represent the Raman frequency shifts obtained when the time intervals between the two pulses are different.

[0053] In some embodiments, when the first light beam is pump light and the second light beam is Stokes light, the cutoff wavelength of the first dichroic mirror is 950 nm; the cutoff wavelength of the second dichroic mirror is 680 nm; the center wavelength of the filter is 650 nm, and the bandwidth of the filter is 40 nm.

[0054] In this embodiment, two laser beams are combined by a dichroic mirror with a cutoff wavelength of 950nm and, after strict collinear adjustment, enter the scanner. They form an object-side telecentric optical path through the scanning lens and tube lens, and are then focused on the sample surface by the microscope objective. The CARS signal generated by the sample is collected by the same objective lens, separated and purified by a dichroic mirror with a cutoff wavelength of 680nm and a bandpass filter with a center wavelength of 650nm and a bandwidth of 40nm. After that, it is focused by a lens onto a photomultiplier tube for detection. Finally, the acquisition software uses the intensity information obtained by point-by-point scanning to generate a CARS image.

[0055] In some embodiments, when the first light beam is Stokes light and the second light beam is pump light, the cutoff wavelength of the first dichroic mirror needs to be adjusted according to actual conditions.

[0056] In some embodiments, the half-wave plate and the quarter-wave plate are respectively fixed on a flip mirror frame.

[0057] Specifically, in order to facilitate the introduction and withdrawal of the half-wave plate and the 1 / 4 wave plate, the half-wave plate and the 1 / 4 wave plate are respectively installed on a 90-degree flip frame. When the wave plate is needed, the frame is flipped to a vertical position, and when it is not needed, the frame only needs to be laid flat and reset.

[0058] In some embodiments, the central wavelength of the pump light is 800 nm; the central wavelength of the Stokes light is 1040 nm; the Raman frequency shift detected by the spectral microscopy imaging system is in the range of 2735-3035 cm -1 between.

[0059] In some embodiments, the first reflector group, the second reflector group, the third reflector group, and the fourth reflector group may all be composed of a plurality of reflectors for changing the optical path.

[0060] In this embodiment, the first reflector group and the fourth reflector group each consist of one reflector; the second reflector group consists of three reflectors; and the third reflector group consists of two reflectors.

[0061] In addition, Figure 4 As shown, the present invention also provides a method for measuring the unsaturation distribution of oil based on a spectral microscopy imaging system, comprising the steps of:

[0062] Step S10: modulating the pump light and the Stokes light into circularly polarized lights with opposite rotation directions, adjusting the acquisition parameters, and acquiring a CARS image at a specific wavenumber of the oil sample to be tested or a CARS image sequence corresponding to different wavenumbers;

[0063] Step S20: Remove the half-wave plate and the quarter-wave plate in the spectral microscopy imaging system, collect a reference CARS image of pure water at the corresponding wavenumber, and calculate the average value of the image intensity as the calibration reference intensity value;

[0064] Step S30: dividing the intensity value of each pixel in the CARS image or the CARS image sequence by the calibration reference intensity value for calibration, and performing normalization processing to obtain a calibrated CARS image;

[0065] Step S40: analyzing the spectral intensity and image of the corrected CARS image at the specific wavenumber to obtain the unsaturation and distribution of the oil in the oil sample to be tested.

[0066] In this embodiment, a half-wave plate is first introduced into the pump light path, and its fast axis direction is rotated so that the polarization direction of the pump light is perpendicular to that of the Stokes light; then a 1 / 4 wave plate is introduced into the beam combining path, and its fast axis direction is rotated to a position of 45 degrees to the polarization direction of the two beams of light, so that the polarization states of the two beams of light become left-handed and right-handed circularly polarized light, respectively. During the adjustment process, a polarizer and a laser power meter can be used to test the polarization states of the two beams of light; when collecting pure water signals as a reference for intensity correction, the two wave plates only need to be removed to return the two beams of light to their original linearly polarized light; and when collecting data on the sample to be tested for measuring the unsaturation and distribution of oils and fats, the two wave plates need to be flipped up to a vertical position to suppress isotropic NRB.

[0067] Specifically, by introducing a half-wave plate and a quarter-wave plate into the optical path, the two beams required to generate the CARS signal are modulated into circularly polarized light with opposite rotation directions, thereby achieving the effect of removing the NRB effect in the CARS signal. A spectral microscopy system equipped with the half-wave plate and the quarter-wave plate is then used to acquire CARS images of the oil-containing sample at a specific Raman frequency shift or a sequence of CARS images corresponding to different wavenumbers. The half-wave plate and the quarter-wave plate are then removed. While keeping all other acquisition parameters unchanged, CARS images of pure water are acquired at the corresponding wavenumbers using the reference sample, and the average signal intensity is calculated as the reference data for spectral intensity correction. The intensity value of each pixel in the CARS image of the oil sample is then divided by the pure water signal intensity value at the corresponding wavenumber. This corrects the CARS spectral intensity distortion caused by the different degrees of overlap of laser pulses in the time domain during the SF process and obtains accurate CARS spectral intensity. Finally, by analyzing the corrected CARS images at specific wavenumbers, the spectral microscopy system can measure the unsaturation distribution of the oil. This spectral microscopy imaging method for measuring the unsaturation distribution of oils and fats has the advantages of high signal intensity, low background noise, fast detection speed, high resolution, and non-destructiveness. It can not only accurately measure the unsaturation of oils and fats, but also achieve two-dimensional or three-dimensional microscopic imaging to analyze the distribution of unsaturation in the sample in detail; at the same time, it can significantly improve the accuracy and efficiency of oil quality analysis.

[0068] In this embodiment, the calibrated CARS spectral intensity is used to quantitatively analyze the unsaturation and distribution of oils and fats: the intensity values ​​of the CARS image of the sample to be tested obtained after the above processing at different wavenumbers will be able to more accurately reflect the different vibration modes of the sample. Therefore, by analyzing the calibrated CARS image at a specific wavenumber, the unsaturation and distribution of the oil sample can be obtained. The characteristic wavenumbers related to the measurement of oil and fat unsaturation are usually 1660cm -1 and 3011cm -1The former corresponds to the stretching vibration of the C=C bond and is a key indicator for measuring the unsaturation of oils and fats. The latter corresponds to the stretching vibration of the asymmetric olefin=CH bond and is indirectly affected by the presence of the C=C bond. It is also often used to measure the unsaturation of oils and fats. In addition, there is 1750cm -1 , which is related to the stretching vibration of the C=O bond of the carboxylic acid. In this embodiment, after quantitative analysis and correction, 3011 cm -1 CARS image at wavenumbers, or analyze its difference with 2850cm -1 The ratio image of the CARS image at the wavenumber (corresponding to the stretching vibration of the C-H bond) can be used to obtain the unsaturation of the oil and its analysis.

[0069] In some embodiments, the acquisition parameters include laser power, scanning field of view, magnification, and acquisition frame number.

[0070] In this embodiment, the method for measuring the unsaturation distribution of oils and fats based on a spectral microscopy imaging system has the following advantages over existing methods:

[0071] 1) The calibration method for the SF-CARS microscopic imaging spectral intensity of the present invention is to first suppress the influence of isotropic NRB in the sample by circular polarization modulation, and then use the NRB signal of pure water obtained under non-circular polarization modulation conditions to characterize the degree of overlap of the two laser pulses in the SF-CARS in the time domain, thereby using the division method to correct the influence of the pulse time domain overlap on the spectral intensity.

[0072] 2) The unsaturation and distribution of oil in the sample are measured using calibrated CARS microscopy images. -1 ) has a positive correlation with the unsaturation of oil. The calibrated spectral intensity can be used to characterize the degree of oil unsaturation, and the calibrated image can intuitively reflect the distribution of oil unsaturation.

[0073] In some embodiments, the position of the precision translation stage in the time delay module is continuously adjusted (one position corresponds to a specific wavenumber and is calibrated in advance with a standard sample), and a CARS image is acquired at each position. The average intensity of a region with uniform intensity in the image is calculated, and a normalized curve of the average intensity versus the corresponding wavenumber is plotted to obtain the CARS spectrum.

[0074] As an application example, taking eicosapentaenoic acid methyl ester (EPA ME) as a sample, the CARS spectra of eicosapentaenoic acid methyl ester obtained by the spectral microscopy imaging system under different conditions are as follows: Figure 5As shown in Figure 2, EPA itself is an ω-3 unsaturated fatty acid containing five C=C bonds. Methyl esterification improves its solubility and stability, but its biological activity still depends mainly on the structure of EPA. The spontaneous Raman spectrum of EPA ME shows that it has a wavelength of 3011 cm -1 There is a significant Raman peak at (corresponding to the stretching vibration of the =CH bond, indirectly affected by the C=C bond), and its normalized intensity is about 0.80. Figure 1 The spectral microscopy imaging system shown in FIG2 obtains CARS spectra of EPA ME before circular polarization modulation is introduced (case 1), after circular polarization modulation is introduced to suppress NRB (case 2), and after further correction using pure water signal (case 3). The results are shown in FIG2. Figure 5 It can be seen that without using the method proposed by the present invention, EPA ME at 3011cm -1 The peak at 0.12 was only 0.12. However, after introducing circular polarization modulation to suppress NRB, the peak at this wavenumber was increased to 0.20. After further correction using a pure water signal, the peak intensity was restored to 0.77, close to that of the spontaneous Raman spectrum. These results indicate that when the Raman frequency shift is just at the edge of the detection range, the change in spectral intensity caused by the decrease in pulse time domain overlap during the SF process is the main factor affecting the accuracy of spectral intensity. The pure water signal correction method proposed in this invention can effectively correct this effect, thereby obtaining more accurate spectral intensity and reducing the impact on subsequent quantitative analysis.

[0075] That is, case 1 is that circular polarization modulation is not introduced and pure water signal is not used for intensity correction; case 2 is that circular polarization modulation is introduced to suppress NRB; case 3 is that circular polarization modulation is introduced to suppress NRB and pure water signal is used for intensity correction.

[0076] Alternatively, for pure solution samples (such as EPA ME in the above example), only Figure 5 In this way, the area with relatively uniform signal is selected from the CARS images with different wave numbers to calculate the intensity average value, and the CARS spectrum is made. The corrected characteristic Raman peak (here refers to 3011cm -1 ) to characterize the unsaturation of oils and fats. However, for complex samples with structure or different components, the method of the present invention is more significant in that it can also analyze CARS images at specific Raman peaks to study the distribution of oil unsaturation in the sample and how this distribution changes over time. As a simple example, the imaging results of the edge of an EPA ME sample are used to illustrate. Figure 6 Shown at 3011cm -1The image at , which was collected after the introduction of circular polarization modulation suppression NRB, and the intensity value of each pixel in the image has been spectrally corrected with the pure water signal, so its intensity can be directly used to evaluate the degree of oil unsaturation (that is, the larger the grayscale value corresponding to the intensity, the higher the unsaturation). Figure 6 The EPAME sample is present in the right part of the image, but not in the left part. Therefore, the unsaturation of the oil in the two parts is different. This difference can be directly seen from the intensity distribution of the image, that is, the intensity distribution of the image represents the distribution of the unsaturation of the oil. If the sample contains different structures or components, the unsaturation distribution of the oil in the sample can be analyzed by analyzing the intensity distribution of the image. Furthermore, if the specific value of the unsaturation of the oil is to be quantitatively calculated, the method of the present invention can be used to measure several standard samples of known unsaturation, and the 3011cm -1 The quantitative relationship between image intensity and unsaturation is calibrated. For an image of an unknown sample, the calibration results can be used to convert the intensity value of each pixel into an unsaturation value and display it in pseudo-color, thereby obtaining an image of the oil unsaturation distribution in the sample. By performing the same processing on images acquired at different time points, the temporal change in the oil unsaturation distribution in the sample can also be determined.

[0077] In summary, the present invention provides a spectral microscopy imaging system and method for measuring the unsaturation distribution of oils and fats. The spectral microscopy imaging system includes: a femtosecond laser for emitting a first light beam and a second light beam that are different from each other, as well as a first optical path component, a second optical path component, a beam combining optical path component, a CARS signal optical path component, and an image generation module; the first light beam is pump light or Stokes light, and the second light beam is Stokes light or pump light. The present invention introduces a half-wave plate and a quarter-wave plate into an optical path to modulate the two beams of light required to generate a CARS signal into circularly polarized light with opposite rotation directions, thereby achieving the effect of removing the NRB effect in the CARS signal. A spectral microscopy system equipped with the half-wave plate and the quarter-wave plate is then used to collect a CARS image of a sample containing oil at a specific Raman frequency shift or a CARS image sequence corresponding to different wavenumbers. The half-wave plate and the quarter-wave plate are then removed, and while keeping all other acquisition parameters unchanged, a CARS image of pure water is collected at a corresponding wavenumber using the reference sample, and the average signal intensity is calculated as reference data for spectral intensity correction. The intensity value of each pixel in the CARS image of the oil sample is then divided by the intensity value of the pure water signal at the corresponding wavenumber, thereby correcting the CARS spectral intensity distortion caused by the different degrees of overlap of laser pulses in the time domain during the SF process and obtaining accurate CARS spectral intensity. Finally, the spectral microscopy system is used to measure the unsaturation distribution of the oil by analyzing the corrected CARS image at the specific wavenumber. In addition, this method has the advantages of high signal intensity, low background noise, fast detection speed, high resolution, and non-destructiveness. It can not only accurately measure the unsaturation of oils and fats, but also realize two-dimensional or three-dimensional microscopic imaging and analyze the distribution of unsaturation in the sample in detail; at the same time, it can significantly improve the accuracy and efficiency of oil quality analysis, providing a reliable new tool for food and other related industries.

[0078] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats, characterized in that: include: A femtosecond laser, configured to emit a first light beam and a second light beam that are different from each other; the first light beam is a pump light or a Stokes light, and the second light beam is a Stokes light or a pump light; The first optical path component includes a first power adjustment module, a first glass rod, a first reflector group, a first beam expander, and a half-wave plate arranged in sequence along the first optical path; The second optical path assembly includes a second power adjustment module, a second glass rod, a second reflector group, a second beam expander, a third reflector group, a time delay module, and a fourth reflector group, which are sequentially arranged along the second optical path; A beam combining optical path assembly, comprising a first dichroic mirror for combining a first light beam passing through the first optical path assembly and a second light beam passing through the second optical path assembly, and a quarter wave plate, a scanner, a scanning lens, a tube lens, and a microscope objective lens sequentially arranged along the beam combining optical path; the microscope objective lens is used to focus the combined light beam on a sample surface and collect a CARS signal generated by the sample; A CARS signal optical path component includes a second dichroic mirror, a filter, a lens, and a photomultiplier tube sequentially arranged along the CARS signal optical path; An image generation module, configured to generate a CARS image from the information obtained by the photomultiplier tube; The first power regulating module is used to adjust the power and polarization direction of the first light beam; the second power regulating module is used to adjust the power and polarization direction of the second light beam; the first glass rod is used to chirp and disperse the first light beam passing through the first power regulating module into picosecond pulse light; the second glass rod is used to chirp and disperse the second light beam passing through the second power regulating module into picosecond pulse light; the first power regulating module and the second power regulating module are both composed of a half-wave plate and a polarization beam splitter.

2. The spectral microscopy imaging system for measuring unsaturation distribution of oils and fats according to claim 1, characterized in that: The time delay module is composed of a precision displacement stage and a pair of reflecting mirrors fixed on the precision displacement stage.

3. The spectral microscopy imaging system for measuring unsaturation distribution of oils and fats according to claim 1, characterized in that: The half-wave plate and the quarter-wave plate are respectively fixed on the flip mirror frame.

4. A method for measuring the unsaturation distribution of oils and fats based on the spectral microscopy imaging system for measuring the unsaturation distribution of oils and fats according to any one of claims 1 to 3, characterized in that: Including steps: The pump light and Stokes light are modulated into circularly polarized lights with opposite rotation directions. After adjusting the acquisition parameters, the CARS image at a specific wavenumber of the oil sample to be tested or the CARS image sequence corresponding to different wavenumbers is acquired; Remove the half-wave plate and quarter-wave plate in the spectral microscopy imaging system, collect the baseline CARS image of pure water at the corresponding wavenumber, and calculate the average value of the image intensity as the calibration baseline intensity value; The intensity value of each pixel in the CARS image or the CARS image sequence is divided by the correction reference intensity value for correction, and normalized to obtain a corrected CARS image; The spectral intensity and image of the corrected CARS image at the specific wavenumber are analyzed to obtain the unsaturation and distribution of the oil in the oil sample to be tested.

5. The method for measuring unsaturation distribution of oils and fats according to claim 4, characterized in that: The acquisition parameters include laser power, scanning field of view, magnification and acquisition frame number.

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