Fluorescence-Raman spectrum combined detection device for measuring plant stress

Abstract

The utility model discloses a fluorescence-Raman spectrum combined detection device for measuring plant stress, and belongs to the field of plant physiological information detection. The device comprises an excitation device; the coaxial light path signal separation module is used for focusing the exciting light to the surface of a plant leaf to be detected, separating a generated Raman signal and a fluorescence signal according to wave bands and then guiding the signals to the corresponding detection modules; the fluorescence detection module is used for acquiring a fluorescence signal and analyzing the fluorescence signal to obtain fluorescence spectrum data; the Raman detection module is used for acquiring Raman signals and analyzing the Raman signals to obtain Raman spectrum data; the data processing module is used for synchronously processing the Raman spectrum data and the fluorescence spectrum data so as to obtain physiological parameter information of the plant leaves to be detected. The device realizes coaxial acquisition and separation of two types of signals through a single excitation light source, simplifies a light path structure, improves detection efficiency and data synchronism, and is very suitable for scenes such as plant health assessment, agricultural monitoring and environmental adaptability analysis.

Description

Technical Field

[0001] This application belongs to the field of plant physiological information detection technology, specifically relating to a fluorescence-Raman spectroscopy combined detection device for measuring plant stress. Background Technology

[0002] Plant physiological information detection is a prerequisite for understanding the laws governing plant life activities, optimizing agricultural production, resource utilization, and ecological protection. Taking plant leaves as an example, as living plant organs, their surfaces have complex microstructures (such as stomata, waxy layers, and hairs), are heterogeneous, fragile, and easily damaged, and their physiological state is constantly changing. Therefore, physical contact must be avoided during detection to minimize unacceptable measurement errors. Furthermore, current plant leaf detection methods primarily rely on Raman and fluorescence spectroscopy. One problem is that the unevenness of the plant leaf surface leads to unstable optical coupling efficiency and poor signal repeatability. Additionally, the strong fluorescence of chlorophyll in plant leaves easily interferes with weak Raman signals, affecting the accuracy of the detection results. Another issue is that both require independent excitation sources and optical systems, resulting in complex system structures, low detection efficiency, and difficulty in achieving simultaneous measurements. Moreover, since Raman and fluorescence signals reflect different physiological indicators of plants, time drift or spatial errors can easily be introduced if data is collected at different times or locations, thus affecting data consistency and analytical accuracy.

[0003] Therefore, there is an urgent need for a simple, non-contact combined detection device that can simultaneously acquire Raman and fluorescence signals. Utility Model Content

[0004] In view of this, the primary objective of this application is to provide a fluorescence-Raman spectroscopy combined detection device for measuring plant stress, which adopts a single excitation light source and coaxial optical path design, simplifies the system structure, simultaneously detects Raman and fluorescence spectra, improves the spatial and temporal consistency of analysis and detection, and enables rapid and accurate plant status assessment.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] One aspect of this application discloses a fluorescence-Raman spectroscopy combined detection device for measuring plant stress, comprising:

[0007] An excitation device configured to emit collimated excitation light;

[0008] A coaxial optical path signal separation module includes a convex lens, a first dichroic mirror, a second dichroic mirror, and a reflecting mirror arranged coaxially. The first dichroic mirror is configured to reflect the excitation light and focus it onto the surface of the plant leaf under test through the convex lens, and transmit the Raman signal and fluorescence signal generated by the excited plant leaf. The second dichroic mirror and the reflecting mirror are configured to separate the Raman signal and fluorescence signal according to wavelength and guide them to the corresponding detection modules.

[0009] A fluorescence detection module, configured to acquire fluorescence signals and analyze them to obtain fluorescence spectral data;

[0010] The Raman detection module is configured to acquire Raman signals and analyze them to obtain Raman spectral data.

[0011] And a data processing module, which is configured to simultaneously process the Raman and fluorescence spectral data to obtain physiological parameter information of the plant leaves to be tested.

[0012] In this application, the excitation device includes a laser and a collimating optical system arranged along the optical path, with the laser connected to a pulse modulator. The laser is an instrument capable of emitting excitation light of a preset wavelength. The specific wavelength can be adjusted or selected according to the experimental purpose and research needs. In some specific examples, the laser is a 470nm laser. The pulse modulator is used to modulate the laser, emitting excitation light, which is then collimated into approximately parallel light by the collimating optical system. It is understood that the laser power is adjustable to adapt to different blade types; as an example, a typical value is 10-50mW. There is no particular limitation on the collimating optical system in this application; a structure well-known in the art can be used. As an example, the collimating optical system consists of a lens group.

[0013] In this application, the optical path can be designed according to the function of the coaxial optical path signal separation module. In a typical example, the coaxial optical path signal separation module includes a convex lens, a first dichroic mirror, a second dichroic mirror, and a reflector arranged along the same optical axis, and also includes a Raman signal optical path and a fluorescence signal optical path. The first dichroic mirror is configured to reflect excitation light and transmit signal light generated after the plant leaf is excited. The second dichroic mirror is configured to reflect Raman signal and transmit fluorescence signal, while the reflector is configured to reflect fluorescence signal; or, the second dichroic mirror is configured to reflect fluorescence signal and transmit Raman signal, while the reflector is configured to reflect Raman signal. It should be noted that in this application, the convex lens, the first dichroic mirror, the second dichroic mirror, and the reflector are arranged along the same optical axis, forming a coaxial excitation and signal acquisition path, which improves signal acquisition efficiency and spatial alignment accuracy. More importantly, this application employs a series-connected dichroic mirror (first dichroic mirror and second dichroic mirror) working in conjunction with a reflecting mirror to perform real-time, band-based physical separation of the returned signal light at the front end of the optical path, and guide it to independent fluorescence signal optical paths and Raman signal optical paths respectively. This suppresses strong fluorescence from entering the Raman channel at the source of the optical path, effectively overcoming the interference of strong fluorescence background on weak Raman signals, and simultaneously obtaining high-precision spectral information.

[0014] Furthermore, the Raman signal optical path includes a first filter and a first converging lens arranged along the optical path, thereby focusing the Raman signal onto the subsequent Raman detection module; the fluorescence signal optical path includes a second filter and a second converging lens arranged along the optical path, thereby focusing the fluorescence signal onto the subsequent fluorescence detection module. The first and second filters are band-stop filters, which are used to effectively shield the excitation light background, suppress stray interference from the excitation light background, and improve the signal-to-noise ratio of the Raman / fluorescence signals. For example, the first filter is configured as a band-stop filter with a cutoff wavelength of 470 nm and an optical density OD value greater than 6, used to effectively shield the excitation light background and improve the signal-to-noise ratio of the Raman signal.

[0015] In this application, the Raman detection module includes a Raman transmission fiber and a Raman spectrometer, wherein the incident end of the Raman transmission fiber is located at the focal point of the first converging lens, and the emitting end is aligned with the incident slit of the Raman spectrometer.

[0016] In this application, the fluorescence detection module includes a fluorescence transmission fiber and a fluorescence spectrometer, wherein the incident end of the fluorescence transmission fiber is located at the focal point of the second converging lens, and the exit end is aligned with the incident slit of the fluorescence spectrometer.

[0017] In this application, the data processing module includes a signal preprocessing module, a feature extraction module, and a parameter calculation module;

[0018] The signal preprocessing module is used to perform Raman background subtraction, fluorescence baseline correction, and noise filtering.

[0019] The parameter calculation module is used to estimate chlorophyll content, plant metabolic status, or stress response indicators based on the processed spectrum.

[0020] It is understood that the physiological parameters of plant leaves in this application include, but are not limited to, chlorophyll content and metabolic activity levels. Furthermore, it should be understood that the data processing module in this application refers to a functional system integrating hardware (such as a computer) and software, which processes, analyzes, and calculates the collected or acquired signals / spectrums using data processing and analysis software and computational methods known in the art; therefore, there are no particular limitations here.

[0021] The parameters of the first and second dichroic mirrors, as well as the first and second filters, can be selected and adapted according to the different plant leaves and the specific optical path design. For example, the first dichroic mirror has a spectral characteristic with a reflection band of 470nm ± 10nm and a transmission band of 500nm or higher, and a reflectivity of not less than 98% for 470nm laser light. The second dichroic mirror has a spectral characteristic with a reflection band of 500-650nm and a transmission band of 650-800nm, used to reflect Raman signals and transmit fluorescence signals, respectively. The cutoff wavelength of the first and second filters is 470nm, and the optical density OD value is greater than 6.

[0022] The beneficial effects of this application are:

[0023] The fluorescence-Raman spectroscopy joint detection device in this application employs a single excitation source and a coaxial optical path separation design, simplifying the system structure and improving the spatial and temporal consistency of Raman and fluorescence signals, thus avoiding errors caused by multi-path, asynchronous acquisition in traditional methods. Simultaneously, based on the coordinated design of a band-sequential dichroic mirror and a reflecting mirror, the returned signal light is efficiently and instantly separated by band at the optical path front end, resulting in a spectrum with higher purity and efficiency, overcoming the severe interference of strong fluorescence background on weak Raman signals. The data processing module supports simultaneous spectral analysis and physiological parameter modeling, enabling rapid and accurate plant status assessment.

[0024] In summary, this device enables rapid, non-destructive in-situ detection of plant leaves, overcoming the severe interference of strong fluorescence background on weak Raman signals. This allows for the high-precision and high-synchronization acquisition of fused spectral information for assessing plant physiological stress. It is suitable for plant science research, crop health monitoring, and ecological environment response analysis, demonstrating good practicality and scalability. Attached Figure Description

[0025] Figure 1 This is a preferred embodiment of the fluorescence-Raman spectroscopy combined detection device in this application.

[0026] In the diagram: 11-Laser, 12-Pulse modulator, 13-Lens group; 21-First dichroic mirror, 22-Second dichroic mirror, 23-Reflector, 24-First filter, 25-First converging lens, 26-Second filter, 27-Second converging lens; 31-Raman transmission fiber, 32-Raman spectrometer; 41-Fluorescence transmission fiber, 42-Fluorescence spectrometer; 51-Main fiber, 52-Data processing module; 6-Convex lens. Detailed Implementation

[0027] The fluorescence-Raman spectroscopy combined detection device provided in this application will be further described in detail below with reference to the accompanying drawings.

[0028] It should be noted that when a component is referred to as "fixed to," "set on," or "mounted to" another component, it can be directly on or indirectly on the other component. However, when a component is referred to as "connected to" or "attached to" another component, it can be directly connected to or indirectly connected to the other component. Furthermore, "connection" generally refers to a method of fixing, and this fixing can be any method of fixing conventional in the art, such as "threaded connection," "riveting," or "welding."

[0029] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed or operated in a specific orientation. Therefore, they should not be construed as limitations on this utility model.

[0030] like Figure 1 As shown, this application discloses a fluorescence-Raman spectroscopy combined detection device for measuring plant stress. This device, based on chlorophyll-induced spectroscopy, enables simultaneous detection of plant leaf fluorescence and Raman spectra, allowing for the acquisition of plant physiological information and rapid, accurate plant status assessment. The device includes an excitation device, a coaxial optical path signal separation module, a Raman spectroscopy detection module, a fluorescence spectroscopy detection module, and a data processing module. The following detailed description of the fluorescence-Raman spectroscopy combined detection device will focus on its different module components.

[0031] Please refer to Figure 1The excitation device is used to emit collimated excitation light with a specific wavelength. In a preferred embodiment, the excitation device includes a laser 11 and a lens group 13, which are arranged along the optical path. The laser 11 is connected to a pulse modulator 12, which is used to adjust the laser output frequency. The lens group 13 forms a collimating optical system. In the embodiments of this application, the excitation device composed of these three components can output collimated excitation light with a wavelength of 470 nm.

[0032] A coaxial optical path signal separation module is positioned within the excitation light path and the signal acquisition path. It serves two purposes: firstly, to guide the excitation light to the surface of the plant leaf under test; and secondly, to separate the excited signal light (including Raman and fluorescence signals) from the plant leaf according to wavelength. In an embodiment of this application, the coaxial optical path signal separation module includes a convex lens 6, a first dichroic mirror 21, a second dichroic mirror 22, and a reflecting mirror 23 arranged along the same optical axis. The first dichroic mirror 21 can reflect excitation light with a wavelength of 470 nm and transmit Raman and fluorescence signals with wavelengths greater than 500 nm. Specifically, the first dichroic mirror 21 guides the collimated excitation light to the convex lens 6, which then focuses it onto the surface of the plant leaf under test. Subsequently, the leaf is excited to generate Raman signals (470-600 nm) and fluorescence signals (680-740 nm), and these signals return via a coaxial path, are collimated by the convex lens 6, and then sequentially pass through the first dichroic mirror 21 and the second dichroic mirror 22. The second dichroic mirror 22 can reflect Raman signals with wavelengths in the range of 500-650 nm and transmit fluorescence signals with wavelengths greater than 650 nm. Therefore, the second dichroic mirror 22 is used to reflect Raman signals and transmit fluorescence signals. The Raman signal enters the Raman signal optical path after being reflected by the second dichroic mirror 22. Figure 1As shown, in the Raman signal optical path, a first filter 24 and a first converging lens 25 are sequentially arranged along the optical path to focus the Raman signal onto the Raman signal detection module. Simultaneously, the fluorescence information transmitted through the second dichroic mirror 22 is reflected by the reflecting mirror 23 into the fluorescence signal optical path. In this path, a second filter 26 and a second converging lens 27 are sequentially arranged along the optical path to focus the fluorescence signal onto the fluorescence signal detection module. In this embodiment, based on the aforementioned optical performance requirements, the first dichroic mirror 21 is selected to have a reflection band of 470nm ± 10nm and a transmission band of 500nm or higher, with a reflectivity of not less than 98% for 470nm excitation light. The second dichroic mirror 22 has a reflection band of 500-650nm and a transmission band of 650-800nm. However, it is understood that, depending on the requirements of the sample to be tested and the band selection, those skilled in the art can set the excitation wavelength and the spectral performance of the first and second dichroic mirrors 21 and 22 as needed. Furthermore, in this application, a first filter 24 and a second filter 26 are respectively provided in the Raman signal optical path and the fluorescence signal optical path. These filters are all band-stop filters, used to effectively shield the excitation light background and improve the signal-to-noise ratio of the Raman or fluorescence signal. Their specific spectral performance or parameters can be selected according to actual detection needs. In this embodiment, the cutoff wavelength of the first filter 24 and the second filter 26 is 470nm, and the optical density OD value is greater than 6.

[0033] The Raman detection module includes a Raman transmission fiber 31 and a Raman spectrometer 32. The incident end of the Raman transmission fiber 31 is located at the focal point of the first converging lens 25, and the exit end is aligned with the incident slit of the Raman spectrometer 32.

[0034] The fluorescence detection module includes a fluorescence transmission fiber 41 and a fluorescence spectrometer 42. The incident end of the fluorescence transmission fiber 41 is located at the focal point of the second converging lens 27, and the exit end is aligned with the incident slit of the fluorescence spectrometer 42.

[0035] As a preferred example, the Raman detection module can also be equipped with a CCD or EMCCD structure, and the fluorescence detection module can also be equipped with an amplification circuit to improve the sensitivity and accuracy of detection.

[0036] The signal output terminals of the Raman spectrometer 32 and the fluorescence spectrometer 42 are connected to the data processing module 52 via the main optical fiber 51. The data processing module 52 can be a computer integrated system or functional unit. Specifically, the data processing module 52 includes a signal preprocessing module, a feature extraction module, and a parameter calculation module. The signal preprocessing module is used to perform Raman background subtraction, fluorescence baseline correction, and noise filtering. The parameter calculation module is used to estimate chlorophyll content, plant metabolic state, or stress response index based on the processed spectrum.

[0037] The working process of the combined detection device in this application is as follows:

[0038] After being modulated by the pulse modulator 12, the laser light is collimated into approximately parallel light by the lens group 13 and then irradiates the surface of the plant leaf along a preset optical axis. The laser power is adjustable, with a typical value of 10-50mW, to adapt to different leaf types.

[0039] The excitation light is focused onto the sample surface after being reflected by the first dichroic mirror 21, and the sample is fixed on a sample stage designed to prevent stray light. After the blade is excited, it generates Raman signals (470-600nm) and fluorescence signals (680-740nm), and these two types of signals return along a coaxial path.

[0040] The signal passes sequentially through the first dichroic mirror 21 and the second dichroic mirror 22. The first dichroic mirror 21 reflects the excitation light and transmits the signal light; the second dichroic mirror 22 reflects the Raman signal and transmits the fluorescence signal. After the excitation residual light is filtered out by the band-stop filter (first filter 24), the Raman signal is focused by the first converging lens 25 into the Raman transmission fiber 31 and connected to the Raman spectrometer 32; the fluorescence signal is redirected by the reflecting mirror 23 and focused by the second converging lens 27 into the fluorescence transmission fiber 41 and connected to the fluorescence spectrometer 42.

[0041] The data processing module 52 is used to simultaneously receive two types of spectral data, perform baseline correction, characteristic peak extraction and ratio analysis, and extract parameters such as chlorophyll content and metabolic activity. The results can be displayed through a graphical interface and support wireless remote uploading.

[0042] It is understood that this device can be scalably configured to different excitation wavelengths or to add machine learning modules to adapt to diverse application scenarios, and is not limited to the specific examples in this application.

[0043] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A fluorescence-Raman spectrum combined detection device for measuring plant stress, characterized in that, include: An excitation device configured to emit collimated excitation light; A coaxial optical path signal separation module includes a convex lens, a first dichroic mirror, a second dichroic mirror, and a reflecting mirror arranged coaxially. The first dichroic mirror is configured to reflect the excitation light and focus it onto the surface of the plant leaf under test through the convex lens, and transmit the Raman signal and fluorescence signal generated by the excited plant leaf. The second dichroic mirror and the reflecting mirror are configured to separate the Raman signal and fluorescence signal according to wavelength and guide them to the corresponding detection modules. A fluorescence detection module, configured to acquire fluorescence signals and analyze them to obtain fluorescence spectral data; The Raman detection module is configured to acquire Raman signals and analyze them to obtain Raman spectral data. And a data processing module, which is configured to simultaneously process the Raman and fluorescence spectral data to obtain physiological parameter information of the plant leaves to be tested.

2. The fluorescence-Raman spectroscopy combined detection apparatus according to claim 1, wherein The excitation device includes a laser and a collimating optical system arranged along the optical path, and the laser is connected to a pulse modulator.

3. The fluorescence-Raman spectroscopy combined detection apparatus according to claim 2, wherein The collimating optical system consists of a lens group.

4. The fluorescence-Raman spectroscopic combined detection apparatus according to claim 1, wherein The coaxial optical path signal separation module further includes a Raman signal optical path and a fluorescence signal optical path; Wherein, the second dichroic mirror is configured to transmit a fluorescence signal and reflect a Raman signal to a Raman signal optical path, while the reflector is configured to reflect a fluorescence signal to a fluorescence signal optical path; or, the second dichroic mirror is configured to transmit a Raman signal and reflect a fluorescence signal to a fluorescence signal optical path, while the reflector is configured to reflect a Raman signal to a Raman signal optical path.

5. The fluorescence-Raman spectroscopy combined detection apparatus according to claim 4, wherein The Raman signal optical path includes a first filter and a first converging lens arranged along the optical path.

6. The fluorescence-Raman spectroscopic combined detection apparatus according to claim 5, wherein The first filter is a band-stop filter with a cutoff wavelength of 470nm and an optical density OD value greater than 6.

7. The fluorescence-Raman spectroscopic combined detection apparatus according to claim 4, wherein The fluorescence signal optical path includes a second filter and a second converging lens arranged along the optical path.

8. The fluorescence-Raman spectroscopy combined detection device as described in claim 1, characterized in that, The fluorescence detection module includes a fluorescence transmission fiber and a fluorescence spectrometer.

9. The fluorescence-Raman spectroscopic combined detection apparatus according to claim 1, wherein The Raman detection module includes a Raman transmission fiber and a Raman spectrometer.

10. The fluorescence-Raman spectroscopic combined detection apparatus according to claim 1, wherein The data processing module includes a signal preprocessing module, a feature extraction module, and a parameter calculation module; The signal preprocessing module is used to perform Raman background subtraction, fluorescence baseline correction, and noise filtering. The parameter calculation module is used to estimate chlorophyll content, plant metabolic status, or stress response indicators based on the processed spectrum.

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