Method for detecting internal temperature of plant body and temperature-sensitive probe

Abstract

The invention discloses a method for detecting temperature in a plant body and a temperature-sensitive probe, and the method comprises the following steps: injecting the temperature-sensitive probe into the plant body to obtain a plant body containing the temperature-sensitive probe. And the temperature-sensitive probe is excited, so that the temperature-sensitive probe emits a light-emitting signal. Light-emitting signals are acquired in a delayed mode, and the temperature in the plant is determined according to the light-emitting signals acquired in the delayed mode. Wherein the time delay is smaller than the luminescence life of the temperature-sensitive probe and larger than the luminescence life of autofluorescence of the plant body. According to the invention, the temperature-sensitive probe is input into the plant body to detect the fluorescence signal, so that the nondestructive detection of the temperature in the plant is realized. Through delayed detection, the influence of autofluorescence of plants is eliminated.

Description

Technical Field

[0001] The present invention relates to the field of temperature measurement. More specifically, the present invention relates to a method for detecting the internal temperature of a plant body and a temperature-sensitive probe. Background Art

[0002] As an important environmental factor, temperature is crucial for the growth and development of plants, as well as the yield and quality of crops. The temperature inside a plant body can characterize the hot and cold degrees of the roots, stems, leaves, flowers, and fruits of the plant. The temperature inside the plant body affects various life activities such as enzymatic reactions, cell membrane movements, and transpiration within the plant, and is an important indicator reflecting the physiological activity status of the plant. Accurately detecting the temperature inside the plant body is the premise and key to studying the temperature adaptability and regulation mechanism of plants, and is of great significance for studying the temperature response law and its molecular mechanism of plants. It also has important practical significance for increasing food production and quality and ensuring food security.

[0003] Existing commonly used temperature measurement technologies such as infrared thermography can only detect the surface temperature of plants and cannot detect the internal temperature of plants. Temperature measurement technologies such as thermocouples, due to directly contacting the measured part, easily change the measured environment and cause damage to the measured part, affecting the accuracy of the temperature measurement result. Due to the lack of effective means for characterizing the temperature inside the plant body, currently scientists can only use the environmental temperature to replace the temperature inside the plant body, which has a large error or even error, seriously affecting the development of the field of plant response to temperature.

[0004] The difficulties in detecting the temperature inside the plant body are as follows: 1) Plants are living systems with highly dynamic physiological activities. The detection of the temperature inside the plant body is preferably non-destructive, and the disturbance to the normal physiological process of the plant should be as small as possible. 2) There are a large number of substances such as proteins and small molecules inside the plant body, and these substances often have autofluorescence, and these fluorescence spectra widely exist in the visible light region and the near-infrared light region. 3) The complex and highly dynamic biochemical environment (pH, ionic strength, etc.) inside the plant body may seriously interfere with non-destructive detection signals such as luminescence.

[0005] Currently, the conventional method for eliminating autofluorescence interference is to perform sequential scanning or spectral scanning. Sequential scanning is ineffective for the situation where the excitation spectrum and the emission spectrum overlap simultaneously. Spectral scanning cannot completely remove the complex autofluorescence of plants. The removal of plant autofluorescence has become a bottleneck for high-quality imaging of traditional fluorescence microscopy.

[0006] In view of this, there is an urgent need to provide a measurement method for non-destructively detecting the internal temperature of a plant body and capable of excluding autofluorescence and interference from pH and ionic strength. Summary of the Invention

[0007] To at least solve the above problems, the present invention provides a method for detecting the internal temperature of a plant body, including: injecting a temperature-sensitive probe into the internal part of the plant body to obtain a plant body containing the temperature-sensitive probe; exciting the temperature-sensitive probe to make the temperature-sensitive probe emit a light signal; collecting the light signal with a time delay to obtain a sample signal; determining the internal temperature of the plant body according to the sample signal; wherein, the time delay is less than the luminescence lifetime of the temperature-sensitive probe and greater than the luminescence lifetime of the autofluorescence of the plant body.

[0008] According to an embodiment of the present invention, collecting the light signal with a time delay includes: setting initial parameters to collect the light signal to generate a real-time image; inputting the real-time image into a discrimination model to obtain a real-time offset; adjusting the initial parameters to make the real-time offset meet a preset threshold; the discrimination model is obtained by the following method: collecting the light signal at multiple different offsets in advance to obtain multiple reference images; constructing a discrimination model including the correspondence between the reference images and the offsets.

[0009] According to an embodiment of the present invention, the light signal includes at least one of luminous intensity, luminescence lifetime, and main peak of luminescence spectrum.

[0010] According to an embodiment of the present invention, the luminescence lifetime of the temperature-sensitive probe is greater than 20 ns.

[0011] According to an embodiment of the present invention, the luminescence lifetime of the temperature-sensitive probe is greater than 100 ns.

[0012] According to an embodiment of the present invention, the time delay is set to be greater than or equal to 10 ns.

[0013] According to an embodiment of the present invention, the time delay is set to 50 ns, 100 ns, 300 ns, or 500 ns.

[0014] According to an embodiment of the present invention, the temperature-sensitive probe is prepared by embedding a luminescent material in a polymer material; the luminescent material is selected from one or more of the following materials: semiconductor quantum dots, carbon quantum dots, rare earth luminescent materials, polymer particles containing fluorescent dyes, silica fluorescent nanoparticles, gold nanoclusters, luminescent gold nanoparticles; wherein, the semiconductor quantum dots are selected from one or more of the following materials: CdSe, CdSe@ZnS, CdSeS, CdSeS@ZnS, CdTe, CdTe@ZnS quantum dots; the rare earth luminescent materials are selected from one or more of the following materials: rare earth complex nanoluminescent materials containing one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), rare earth-doped upconversion nanoparticles; the fluorescent dyes are selected from one or more of the following materials: fluorescein isothiocyanate, rhodamine.

[0015] According to an embodiment of the present invention, the luminescent gold nanoparticles are obtained by a photoreaction, and the particle size ranges from 2 to 100 nm.

[0016] According to an embodiment of the present invention, the luminescent material of the temperature-sensitive probe is selected from one or more of the following materials: erbium-doped ytterbium-doped sodium yttrium fluoride nanoparticles NaYF4,Yb,Er@NaYF4,Yb,Nd encapsulated with neodymium-doped ytterbium-doped sodium yttrium fluoride; or thulium-doped ytterbium-doped sodium yttrium fluoride nanoparticles NaYF4,Yb,Tm@NaYF4,Yb,Nd encapsulated with neodymium-doped ytterbium-doped sodium yttrium fluoride; or erbium-doped ytterbium-doped sodium yttrium fluoride nanoparticles NaYF4:Yb,Er.

[0017] According to an embodiment of the present invention, the temperature-sensitive probe is selected from one or more of the following substances: cadmium sulfide quantum dots CdS / ZnS QDs coated with zinc sulfide; cadmium selenide quantum dots CdSe / ZnS QDs coated with zinc sulfide; cadmium telluride quantum dots CdTe / ZnS QDs coated with zinc sulfide; cadmium selenosulfide quantum dots CdSeS QDs; cadmium tellurosulfide quantum dots CdTeS QDs; cadmium selenosulfide quantum dots CdSeS / ZnS QDs coated with zinc sulfide; cadmium tellurosulfide quantum dots CdTeS / ZnS QDs coated with zinc sulfide; where QDs represents quantum dots. Luminescent gold nanoparticles AuNP@DHLA with dihydrolipoic acid as the surface ligand; luminescent gold nanoparticles AuNP@HTT with 2-hexylthio-1,3,4-thiadiazole-5-thiol as the surface ligand; polymer-coated luminescent gold nanoparticles p-AuNP@DHLA with DHLA as the surface ligand; polymer-coated luminescent gold nanoparticles p-AuNP@HTT with HTT as the surface ligand; europium complex Eu(tta)3dpbt, where Eu represents europium, dpbt represents 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, the English name is 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, and tta represents diacyl trifluoroacetone.

[0018] According to an embodiment of the present invention, the temperature-sensitive probe is prepared by embedding a luminescent material in a polymer material, including: mixing the polymer material and the luminescent material in a tetrahydrofuran solution to obtain a first mixture, dropping the first mixture into ultrapure water, and continuously stirring during the dropping process to obtain a second mixture; allowing the second mixture to stand for 10-15 minutes to obtain a third mixture; removing tetrahydrofuran from the third mixture to obtain a solution containing the temperature-sensitive probe. The polymer material is selected from existing materials that do not conflict with the plant internal environment, such as PMMA-co-MAA copolymers and their analogs.

[0019] According to another aspect of the present invention, there is provided a temperature-sensitive probe for detecting the internal temperature of a plant body, which is prepared by embedding a luminescent material in a polymer material; the luminescent material is selected from one or more of the following materials: semiconductor quantum dots, carbon quantum dots, rare earth luminescent materials, polymer particles containing fluorescent dyes, silica fluorescent nanoparticles, gold nanoclusters, luminescent gold nanoparticles; wherein, the semiconductor quantum dots are selected from one or more of the following materials: CdSe, CdSe@ZnS, CdSeS, CdSeS@ZnS, CdTe, CdTe@ZnS quantum dots; the rare earth luminescent materials are selected from one or more of the following materials: rare earth complex nanoluminescent materials containing one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y); the fluorescent dyes are selected from one or more of the following materials: fluorescein isothiocyanate, rhodamine.

[0020] According to an embodiment of the present invention, the temperature-sensitive probe is selected from one or more of the following substances: CdS@ZnS, CdSe@ZnS, CdTe@ZnS, CdSeS, CdTeS, CdSeS@ZnS, CdTeS@ZnS quantum dots; AuNP@DHLA, AuNP@HTT, p-AuNP@DHLA, p-AuNP@HTT; Eu(tta)3dpbt, wherein Eu represents europium, dpbt represents 2-(N,N-diethylaniline-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, and tta represents diacyl trifluoroacetone.

[0021] In the present invention, by inputting a biocompatible temperature-sensitive probe into the plant body and detecting its luminescence signal, the internal temperature of the plant body is obtained, realizing non-destructive detection. Through delayed detection, the influence of the autofluorescence of the plant body is filtered out. By selecting the temperature-sensitive probe in the embodiment of the present invention, the interference of factors such as pH and ionic strength can be excluded, and accurate internal temperature measurement results can be obtained. Description of the Drawings

[0022] Figure 1 Shows a schematic diagram of the steps for detecting the internal temperature of a plant body according to an embodiment of the present invention;

[0023] Figure 2 Shows a schematic diagram of an imaging objective lens collecting a luminescence signal to generate an image according to an embodiment of the present invention;

[0024] Figure 3Schematic diagram showing the photoluminescence spectrum of p-AuNP@HTT according to an embodiment of the present invention;

[0025] Figure 4 Transmission electron microscopy image showing p-AuNP@HTT according to an embodiment of the present invention;

[0026] Figure 5 Statistical histogram showing the particle size distribution of p-AuNP@HTT according to an embodiment of the present invention;

[0027] Figure 6 Schematic diagram showing the change of the luminescence spectrum of p-AuNP@HTT with temperature according to an embodiment of the present invention;

[0028] Figure 7 Schematic diagram showing the change of the luminescence intensity at the emission peak of p-AuNP@HTT with temperature according to an embodiment of the present invention;

[0029] Figure 8 Schematic diagram showing the change of the luminescence intensity of p-AuNP@HTT with temperature under delayed acquisition according to an embodiment of the present invention;

[0030] Figure 9 Schematic diagram showing the comparison of the temperature detected based on p-AuNP@HTT and the temperature detected based on a thermocouple under delayed acquisition according to an embodiment of the present invention;

[0031] Figure 10 Schematic diagram showing the luminescence lifetime of p-AuNP@HTT according to an embodiment of the present invention;

[0032] Figure 11 Schematic diagram showing the change of the luminescence lifetime of p-AuNP@HTT with temperature according to an embodiment of the present invention;

[0033] Figure 12 Schematic diagram showing the change of the luminescence intensity of p-AuNP@HTT in the plant physiological pH range according to an embodiment of the present invention;

[0034] Figure 13 Schematic diagram showing the change of the luminescence intensity of p-AuNP@HTT with Ca 2+ concentration according to an embodiment of the present invention;

[0035] Figure 14 Schematic diagram showing the delayed imaging of HeLa cells labeled with p-AuNP@HTT according to an embodiment of the present invention;

[0036] Figure 15 Schematic diagram showing the change of the luminescence intensity of the nano-luminescence probe inside tobacco with the ambient temperature under high temperature stress according to an embodiment of the present invention;

[0037] Figure 16 The schematic diagram shows the corresponding relationship between the internal temperature change amount and the environmental temperature change amount of tobacco under high temperature stress according to an embodiment of the present invention;

[0038] Figure 17 The schematic diagram shows the change of the luminescence intensity of the nano-luminescence probe inside tobacco with the environmental temperature under low temperature stress according to an embodiment of the present invention;

[0039] Figure 18 The schematic diagram shows the corresponding relationship between the internal temperature change amount and the environmental temperature change amount of tobacco under low temperature stress according to an embodiment of the present invention;

[0040] Figure 19 The schematic diagram shows the change of the luminescence intensity of the nano-luminescence probe inside tomato with the environmental temperature under low temperature stress according to an embodiment of the present invention;

[0041] Figure 20 The schematic diagram shows the corresponding relationship between the internal temperature change amount and the environmental temperature change amount of tomato under low temperature stress according to an embodiment of the present invention;

[0042] Figure 21 The schematic diagram shows the trend of the luminescence intensity of p-AuNP@DHLA changing with the environmental temperature according to an embodiment of the present invention;

[0043] Figure 22 The schematic diagram shows the change of the luminescence intensity of p-CdSeS QDs with the environmental temperature according to an embodiment of the present invention;

[0044] Figure 23 The schematic diagram shows the change of the luminescence intensity of the nano-luminescence probe inside tobacco with the environmental temperature under high temperature stress according to an embodiment of the present invention;

[0045] Figure 24 The schematic diagram shows the corresponding relationship between the internal temperature change amount and the environmental temperature change amount of tobacco under high temperature stress according to an embodiment of the present invention. Detailed implementation manners

[0046] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0047] Next, the specific implementation manners of the present invention will be described in detail in conjunction with the accompanying drawings.

[0048] Figure 1 The schematic diagram shows the steps for detecting the internal temperature of a plant body according to an embodiment of the present invention.

[0049] As Figure 1 shown, method 100 for detecting the internal temperature of a plant body includes step S101 of injecting a temperature-sensitive probe into the interior of the plant body to obtain a plant body containing the temperature-sensitive probe; step S102 of exciting the temperature-sensitive probe to cause the temperature-sensitive probe to emit a light signal; step S103 of collecting the light signal with a time delay to obtain a sample signal; and step S104 of determining the internal temperature of the plant body based on the sample signal, wherein the time delay is less than the luminescence lifetime of the temperature-sensitive probe and greater than the luminescence lifetime of the autofluorescence of the plant body.

[0050] A relatively common method for detecting the internal temperature of a plant body is to inject a temperature-sensitive probe into the plant body and then excite the temperature-sensitive probe, and determine the internal temperature of the plant body by detecting the light signal emitted by the temperature-sensitive probe.

[0051] The inventors recognized that in addition to different spectral parameters, different types of fluorescence also have differences in luminescence lifetime. For example, the luminescence lifetime of the autofluorescence of a plant body is usually short, generally less than 10 ns. Therefore, when performing luminescence detection, it is possible to consider separating the luminescence signal from the time dimension to remove the interference of background noises such as autofluorescence, reflected light, and stray light.

[0052] Table 1 shows the luminescence lifetime table of some luminescent materials.

[0053] Table 1.

[0054] Name of the luminescent material Luminescence lifetime Fluorescein 4.3 ns Autofluorescence of plants <10 ns Quantum dots (QDs) 26.2 - 160 ns Green fluorescent protein (GFP) 2.8 ns Rare earth luminescent materials 62 - 820 μs Gold nanoclusters (AuNCs) >100 ns Luminescent gold nanoparticles (L - AuNPs) 10 - 720000 ns

[0055] As can be seen from Table 1, the luminescence lifetime of the autofluorescence of the plant body is short. To remove the interference of autofluorescence, the temperature-sensitive probe is prepared from a luminescent material with a luminescence lifetime greater than that of the autofluorescence of the plant body. In this article, the luminescence lifetime of the temperature-sensitive probe refers to the luminescence lifetime of the luminescent material contained therein.

[0056] Specifically, a pulsed laser is used in combination with a long-lifetime fluorescent probe. The fluorescent probe is injected into the plant body. When the fluorescent substance in the fluorescent probe is excited, the fluorescent signal is not collected immediately, but instead, after waiting for the fluorescence to decay for a period of time (such as 20 ns or 50 ns) until the background noises such as autofluorescence are completely decayed, the luminescence signal of the long-lifetime fluorescent probe is collected. This technique is called time-gated fluorescence imaging technology (Time-gated fluorescenceim aging). By not detecting the short-lifetime signals but only detecting the long-lifetime signals, it is possible to obtain an image of long-lifetime fluorescent (or luminescent) substances with high contrast. By using multi-period signals to increase the signal-to-noise ratio, the imaging quality can be improved and the interference of short-lifetime background fluorescence (or other luminescence) on the image can be reduced.

[0057] Based on this, in an embodiment of the present invention, the selection criteria for the temperature-sensitive probe are as follows: the luminescence lifetime of the temperature-sensitive probe is greater than the lifetime of the autofluorescence of the plant body. For example, the luminescence lifetime of the selected temperature-sensitive probe is greater than 100 ns.

[0058] According to an embodiment of the present invention, the nano-luminescent materials for preparing the temperature-sensitive probe include, but are not limited to, semiconductor quantum dots, carbon quantum dots, rare-earth luminescent materials, fluorescent nanoparticles containing traditional fluorescent dyes such as FITC, silica fluorescent nanoparticles, gold nanoclusters, luminescent gold nanoparticles, etc. There is a uniquely determined linear or non-linear one-to-one correspondence between the luminescence signal of such nano-luminescent materials and temperature.

[0059] Among them, the semiconductor quantum dots include, but are not limited to, quantum dots such as CdSe, CdSe@ZnS, CdSeS, CdSeS@ZnS, CdTe, CdTe@ZnS, etc. @ represents a core-shell structure, with the former being the core and the latter being the shell.

[0060] The rare-earth luminescent materials include, but are not limited to, rare-earth complex nano-luminescent materials and rare-earth doped upconversion nanoparticles containing lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), etc. For example, Eu(tta)3dpbt, sodium yttrium fluoride doped with neodymium and ytterbium and coated with sodium yttrium fluoride doped with erbium and ytterbium, NaYF4, Yb, Er@NaYF4, Yb, Nd; or sodium yttrium fluoride doped with neodymium and ytterbium and coated with sodium yttrium fluoride doped with thulium and ytterbium, NaYF4, Yb, Tm@NaYF4, Yb, Nd; or sodium yttrium fluoride doped with erbium and ytterbium, NaYF4:Yb, Er.

[0061] Gold nanoclusters refer to gold nanoclusters with a size < 2 nm and luminescence properties.

[0062] Luminescent gold nanoparticles (L-AuNPs) refer to gold nanoparticles with a size > 2 nm and luminescence properties. Such substances can be screened and obtained from gold nanoparticles through existing technologies. For example, the luminescence intensity test is carried out in plants, which will not be elaborated in the present invention. In particular, the size range of the luminescent gold nanoparticles is 2 nm - 100 nm.

[0063] For example, AuNP@HTT represents a luminescent gold nanoparticle with a surface ligand (ligand) of HTT. Among them, @ represents a core-shell structure, with the former being the core and the latter being the shell.

[0064] For another example, p-AuNP@HTT (polymer encapsulated AuNC@HTT) represents a luminescent gold nanoparticle with a polymer coating and a surface ligand of HTT. Herein, HTT represents 2-n-hexylthio-1,3,4-thiadiazole-5-thiol.

[0065] Similarly, p-AuNP@DHLA represents a luminescent gold nanoparticle with a polymer coating and a surface ligand of DHLA. Herein, DHLA represents dihydrolipoic acid.

[0066] Specifically, the luminescent material of the thermosensitive probe is selected from: cadmium sulfide quantum dots CdS / ZnS QDs encapsulated with zinc sulfide; cadmium selenide quantum dots CdSe / ZnS QDs encapsulated with zinc sulfide; cadmium telluride CdTe / ZnS QDs encapsulated with zinc sulfide; cadmium selenosulfide quantum dots CdSeS QDs; cadmium tellurosulfide quantum dots CdTeS QDs; cadmium selenosulfide quantum dots CdSeS / ZnS QDs encapsulated with zinc sulfide; cadmium tellurosulfide quantum dots CdTeS / ZnS QDs encapsulated with zinc sulfide; wherein, QDs represents quantum dots. Luminescent gold nanoparticles AuNP@HTT with a surface ligand of 2-n-hexylthio-1,3,4-thiadiazole-5-thiol; luminescent gold nanoparticles AuNP@DHLA with a surface ligand of dihydrolipoic acid; p-AuNP@DHLA, a luminescent gold nanoparticle with a polymer coating and a surface ligand of DHLA; p-AuNP@HTT, a luminescent gold nanoparticle with a polymer coating and a surface ligand of HTT; europium complex Eu(tta)3dpbt, wherein dpbt represents 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, English name 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, and tta represents thenoyltrifluoroacetonato, English name thenoyltrifluoroacetonato.

[0067] In step S101, after injecting the thermosensitive probe into the interior of the plant body, it needs to be cultured for a period of time, such as 7-10 hours. Preferably, it is cultured for 8 hours to enable the thermosensitive probe to be distributed in the plant to be measured.

[0068] Then, enter step S102, and the excitation light excites the thermosensitive probe in the plant body, causing the thermosensitive probe inside the plant body to emit a luminescent signal. According to an embodiment of the present invention, the luminescent signal includes luminescence intensity, luminescence lifetime, and the main peak of the luminescence spectrum.

[0069] In the present invention, existing excitation devices and acquisition devices can be adopted, such as a laser confocal microscope. The excitation light can be pulsed laser, and the wavelength can be set according to the type of the temperature-sensitive probe. The types of the luminescence signals emitted by the whole plant will change with the passage of time. For example, during the period from the initial moment to the end of the luminescence lifetime of the autofluorescence of the plant, the luminescence signals include the autofluorescence of the plant and the light emitted by the temperature-sensitive probe, and the two are mixed together and difficult to distinguish. When the time exceeds the luminescence lifetime of the autofluorescence of the plant, the autofluorescence of the plant decays, and only the fluorescence of the temperature-sensitive probe with a luminescence lifetime longer than that of the autofluorescence of the plant remains in the luminescence signals.

[0070] Based on this, in step S103, the luminescence signal is collected with a time delay. By collecting the luminescence signal with a time delay, the interference of the autofluorescence of the plant can be filtered out. The time length of the time delay is set to be greater than or equal to the luminescence lifetime of the autofluorescence of the plant and less than the luminescence lifetime of the temperature-sensitive probe. For example, the length of the time delay is set to be time delay ≥ 10 ns. Specifically, the time delay is set to be 50 ns, 100 ns, 300 ns or 500 ns.

[0071] In step S103, collecting the luminescence signal with a time delay includes: setting initial parameters to collect the luminescence signal to generate a real-time image; inputting the real-time image into a discrimination model to obtain a real-time offset; adjusting the initial parameters to make the real-time offset meet a preset threshold; the discrimination model is obtained by the following method: collecting the luminescence signal at multiple different offsets in advance to obtain multiple reference images; constructing a discrimination model including the corresponding relationship between the reference images and the offsets.

[0072] The discrimination model refers to a model constructed in advance for judging the offset of an image. For example, it can be implemented by an artificial neural network. Hereinafter, taking the Z-axis drift of the offset as an example for illustration.

[0073] Figure 2 The schematic diagram of an imaging objective lens collecting a luminescence signal to generate an image according to an embodiment of the present invention is shown.

[0074] As Figure 2As shown, in the imaging system 200, during the imaging process, the imaging objective lens 201 establishes a Cartesian coordinate system, where the plane where the X and Y axes are located is parallel to the focal plane of the imaging objective lens, and the optical signal acquisition direction of the imaging objective lens is the Z axis. Z-axis drift refers to the instability or drift of the focal plane position during the imaging process, which may be caused by factors such as environmental temperature changes, mechanical vibrations, and sample movements. In the embodiments of the present invention, it is mainly caused by changes in the environmental temperature. In the embodiments of the present invention, in order to obtain accurate temperature measurement results, it is necessary to ensure the stability of the focal plane of the imaging objective lens during the process of collecting luminescent signals and imaging.

[0075] First, before starting to detect the internal temperature of the plant body, through z-axis scanning, that is, adjusting different drift distances along the z-axis, multiple different images can be obtained, and these images contain specific markers. For example, image 210, image 220, and image 230.

[0076] Then, select the image 220 with the best imaging effect from the above images, that is, usually the standard image located in the focal plane.

[0077] Then, extract the feature points of the markers on each image and match them with the corresponding feature points of the markers in the standard image, and calculate the Z-axis offset of each image. These offsets describe the displacement of each plane relative to the focal plane.

[0078] Use the above series of data pairs of images and Z-axis offsets as training samples and test samples to train the neural network to obtain a discrimination model.

[0079] When measuring the internal temperature of the plant body, first collect the luminescent signal according to the preset initial parameters, input the obtained real-time image into the discrimination model, and the output result of the discrimination model is the real-time offset of the real-time image. Feed the real-time offset back to the Z-axis adjustment system of the imaging objective lens to adjust the distance of the Z-axis offset, so as to achieve focus tracking imaging.

[0080] Constructing a discrimination model through a neural network is only one implementation method. According to the embodiments of the present invention, when discriminating the offset of the real-time image, it is also possible to calculate the z-axis offset by aligning the real-time image with the sequence images and through interpolation.

[0081] Finally, enter step S104 to determine the internal temperature of the plant body according to the luminescent signal collected with a time delay. According to the corresponding relationship between the change of the luminescent signal of the temperature-sensitive probe and the temperature, the internal temperature of the plant body can be directly obtained.

[0082] In the research on temperature-sensitive materials, the inventors found that gold nanostructures have excellent biocompatibility, are easy to synthesize, have adjustable sizes, and unique physical and chemical properties. Gold nanostructures with luminescent properties usually have a particle size of less than 2 nm, and such nanomaterials are also called gold nanoclusters (AuNCs).

[0083] Gold nanostructures with a size larger than 2 nm, also called gold nanoparticles (AuNPs), have a particle size range of 2 - 100 nm. Due to the presence of surface plasmon resonance (SPR) on their surfaces, almost no photoluminescence properties have been observed. However, with the development of technology and in-depth research, it has been found that some gold nanoparticles with a size larger than 2 nm can also have photoluminescence properties. Such gold nanoparticles with photoluminescence properties are called luminescent gold nanoparticles, that is, L-AuNPs. The screening of luminescent gold nanoparticles can be obtained through a simple photoluminescence test, which will not be elaborated in this invention. For example: The existing patent documents CN113563872A "Hydrophobic Gold Nanoclusters Protected by Thiadiazole Derivatives and Their Photochemical Synthesis Methods" and CN113604215A "Photochemical Synthesis Methods of Gold Nanoclusters Protected by Thiadiazole Derivatives and Their Applications" record the preparation of L-AuNPs by the photochemical method. CN113563872A "Hydrophobic Gold Nanoclusters Protected by Thiadiazole Derivatives and Their Photochemical Synthesis Methods" also records that the prepared L-AuNPs have two-photon luminescence properties and fluorescence lifetimes (τ1 = 0.27 μs, τ2 = 1.21 μs).

[0084] According to an embodiment of the present invention, the luminescent gold nanoparticles are obtained through a photochemical reaction, and the particle size range is 2 - 100 nm. Gold nanoparticles obtained through a photochemical reaction generally have photoluminescence properties.

[0085] According to an embodiment of the present invention, the luminescent material of the temperature-sensitive probe is preferably gold nanoclusters or luminescent gold nanoparticles. For example, the temperature-sensitive probe is p-AuNP@HTT, and the delay is 100 ns. The luminescent nanomaterials of gold nanoclusters or luminescent gold nanoparticles also have strong pH stability and anti-ion interference characteristics, which can make the measurement results more accurate.

[0086] According to an embodiment of the present invention, the temperature detection range in plants is 0 - 60 °C

[0087] According to an embodiment of the present invention, the temperature-sensitive probe p-AuNP@HTT is prepared by the following method: Mix a tetrahydrofuran solution of PMMA-co-MAA copolymer (polymethyl methacrylate-methyl methacrylic acid copolymer) and a tetrahydrofuran solution of AuNP@HTT to obtain a first mixture; at 37 °C, continuously stir ultrapure water, and dropwise add the first mixture into the ultrapure water to obtain a second mixture. React the second mixture for 10 minutes to obtain a third mixture; Step four: at 45 °C, rotary evaporate the third mixture under vacuum to remove tetrahydrofuran, and obtain a solution containing the temperature-sensitive probe p-AuNP@HTT.

[0088] In the present invention, a method for non-destructively detecting a plant body is achieved by inputting a temperature-sensitive probe into the plant body to detect a luminescence signal and obtain the internal temperature. By delaying the detection, the influence of the autofluorescence of the plant body is filtered out.

[0089] In Examples 1 to 10, the temperature-sensitive probe p-AuNP@HTT is taken as an example for illustration.

[0090] In Examples 11 to 12, the temperature-sensitive probe p-AuNP@DHLA is taken as an example for illustration.

[0091] In Examples 13 to 14, the temperature-sensitive probe p-CdSeS QDs is taken as an example for illustration.

[0092] In Example 15, the temperature-sensitive probe p-EuNPs is taken as an example for illustration.

[0093] Example 1.

[0094] Photochemically synthesize luminescent gold nanoparticles (AuNP@HTT) with HTT as a ligand:

[0095] First, prepare an HAuCl4 (10 mM) ethanol solution (Solution A), an NaOH ethanol solution (0.2 M) (Solution B), and an HTT chloroform solution (20 μmol / L) (Solution C) respectively. At 25 °C, mix Solution A (1.0 mL), Solution B (200 μL), and Solution C (9.0 mL), and magnetically stir (100 rpm) for 15 minutes;

[0096] Then, irradiate with an ultraviolet lamp (390 nm, 8 W) for 60 hours to reduce Au and obtain a reaction mixture;

[0097] Then, add the above reaction mixture to cold ethanol at -20 °C and store it in a refrigerator (4 °C) until no yellow precipitate is formed;

[0098] Finally, the precipitate was collected by centrifugation at 4000 rpm for 15 minutes and washed three times with absolute ethanol to obtain the final product AuNP@HTT.

[0099] Example 2.

[0100] Preparation of AuNP@HTT nanoparticles with a polymer cladding (p-AuNP@HTT):

[0101] Polymer material selection: polymethyl methacrylate-methacrylic acid copolymer (PMMA-co-MAA copolymer, Polymethylmethacrylate-co-methacrylic acid).

[0102] First, 1.0 mL of a solution of PMMA-co-MAA copolymer in tetrahydrofuran (concentration 1.0 mg / mL) was mixed with 1.0 mL of a solution of AuNP@HTT in tetrahydrofuran (concentration 1.0 g / mL, prepared in Example 1).

[0103] Then, it was slowly added dropwise to 20 mL of ultrapure water (18 MΩ) at 37 °C, with continuous stirring during the dropping process.

[0104] Then, the reaction was allowed to stand for 10 minutes.

[0105] Finally, tetrahydrofuran was removed by a vacuum rotary evaporator at 45 °C to obtain a solution containing the thermosensitive probe p-AuNP@HTT.

[0106] Example 3.

[0107] Measurement of the emission spectrum of p-AuNP@HTT:

[0108] Figure 3 The schematic diagram of the photoluminescence spectrum of p-AuNP@HTT is shown. The abscissa is the wavelength, with the unit of nanometer (nm), and the ordinate is the photoluminescence intensity (dimensionless relative quantity). The measurement temperature is 25 °C.

[0109] As Figure 3 shown, for the photoluminescence spectrum of p-AuNP@HTT, the maximum emission wavelength is located at 543 nm, and the full width at half maximum is 60 nm, showing good photoluminescence properties.

[0110] Example 4.

[0111] Measurement of the particle size of p-AuNP@HTT:

[0112] The p-AuNP@HTT solution with a concentration of 0.01 mg / mL was dropped onto a copper ultramicrogrid film, dried naturally in the dark, imaged using a TECNAI F30 field emission transmission electron microscope, and the particle size distribution of p-AuNP@HTT was statistically analyzed.

[0113] Figure 4 Figure 4 shows the transmission electron microscope image of p-AuNP@HTT, and the scale bar is 50 nm.

[0114] Figure 5 Figure 5 shows the statistical histogram of the particle distribution of p-AuNP@HTT, and the curve is the Gaussian distribution (Gauss Fit).

[0115] As shown in Figure 4 and Figure 5 As shown, the particle size of p-AuNP@HTT is 24.15 ± 0.46 nm (N = 300). The particle morphology distribution is uniform and the crystal structure is clear and complete. The particle size of the p-AuNP@HTT of the present invention is larger than that of traditional gold nanoclusters (the particle size of gold nanoclusters < 2 nm).

[0116] Example 5.

[0117] Measure the temperature sensitivity of p-AuNP@HTT:

[0118] Figure 6 Figure 6 shows the schematic diagram of the change in the photoluminescence intensity of p-AuNP@HTT with temperature. The abscissa is the wavelength in nanometers (nm), and the ordinate is the photoluminescence intensity. Different curves represent the change in the photoluminescence intensity of p-AuNP@HTT at different temperatures in the range from 5 °C to 60 °C, with a temperature gradient of 5 °C.

[0119] Figure 7 Figure 7 shows the schematic diagram of the change in the relative luminescence intensity at the emission peak of p-AuNP@HTT with temperature. The abscissa is the temperature in °C, and the ordinate is the relative luminescence intensity (dimensionless relative quantity).

[0120] Figure 8 Figure 8 shows the schematic diagram of the change in the relative luminescence intensity of p-AuNP@HTT with the ambient temperature under the condition of delayed acquisition. The abscissa is the ambient temperature in °C, and the ordinate is the relative luminescence intensity (dimensionless relative quantity).

[0121] Figure 9 Figure 9 shows the comparison schematic diagram between the calibrated temperature (the temperature detected by the probe) based on the luminescence intensity detected by p-AuNP@HTT and the ambient temperature detected by the thermocouple under the condition of delayed acquisition.

[0122] As shown in Figure 6As shown, the curves from top to bottom represent the luminescence intensity curves at temperatures ranging from 5°C to 60°C. From the overall trend of the luminescence intensity of p-AuNP@HTT changing with temperature, as the temperature increases from 5°C to 60°C, the luminescence peak of the temperature-sensitive probe p-AuNP@HTT does not shift significantly and is located between 537 - 550 nm, but its luminescence intensity decreases correspondingly with the temperature rise process.

[0123] As Figure 7 shown, the fitting curve of the relative luminescence intensity of p-AuNP@HTT changing with temperature, the fitting function is y = -1.22x + 104.42, and the correlation coefficient between the relative luminescence intensity and temperature is R 2 = 0.994. As the temperature increases from 5°C to 60°C, the luminescence intensity of p-AuNP@HTT gradually decreases and shows a linear relationship: for every 1°C increase in temperature, the luminescence intensity decreases by 1.22%. It can be seen that the luminescence intensity of p-AuNP@HTT has good temperature sensitivity.

[0124] As Figure 8 shown, the straight line is the reference line, and the curve is the relationship curve of the relative luminescence intensity of p-AuNP@HTT changing with the ambient temperature in the delayed acquisition mode. The fitting function is y = -1.23x + 130.92, and the correlation coefficient between the relative luminescence intensity and the ambient temperature is R 2 = 0.993. In the delayed acquisition mode, after a delay of 100 ns, as the temperature increases from 25°C to 50°C, with the increase of the ambient temperature, the relative luminescence intensity of p-AuNP@HTT gradually decreases, and there is a good linear relationship between the decrease amplitude and the increase amplitude of the ambient temperature: for every 1°C increase in the ambient temperature, the luminescence intensity of p-AuNP@HTT decreases by 1.23%. The method of delayed acquisition can be used to obtain the linearly correlated stage of the two, making p-AuNP@HTT suitable for time-gated imaging to detect temperature and accurately detect temperature.

[0125] As Figure 9 shown, the temperature calibrated based on the luminescence intensity detected by p-AuNP@HTT is represented by the curve without floating points, and the ambient temperature detected by the thermocouple is represented by the curve with floating points. The abscissa is time in s, the left ordinate is the ambient temperature in °C, and the right ordinate is the temperature calibrated by luminescence intensity in °C (the temperature detected by the probe). Comparing the two, they are basically the same.

[0126] Example 6.

[0127] Measure the luminescence lifetime of p-AuNP@HTT:

[0128] Figure 10 Shows a schematic diagram of the luminescence lifetime of p-AuNP@HTT. The abscissa is time in ns, and the ordinate is the photoluminescence intensity (normalized, dimensionless).

[0129] As shown Figure 10 in the figure, the luminescence lifetime curve of p-AuNP@HTT at 25 °C. After triple-exponential fitting, its luminescence lifetimes are τ1 = 38.56 ns (5.80%); τ2 = 295.81 ns (25.25%); τ3 = 1472.71 ns (68.95%), and the average luminescence lifetime is 1092.36 ns. Thus, it can be seen that the luminescence lifetime of p-AuNP@HTT is much greater than the lifetime of plant autofluorescence, and time-gated imaging can be used to remove the interference of plant autofluorescence.

[0130] Figure 11 The figure shows the schematic diagram of the luminescence lifetime of p-AuNP@HTT varying with temperature. The abscissa is the temperature in °C, and the ordinate is the luminescence lifetime (fluorescence lifetime) in ns.

[0131] Figure 11 is the fitting curve of the luminescence lifetime (fluorescence lifetime) of p-AuNP@HTT varying with temperature. The fitting function is y = -18.76x + 1743.30, and the correlation coefficient between the luminescence lifetime of p-AuNP@HTT and temperature is R 2 = 0.993. When the temperature increases from 20 °C to 45 °C, the luminescence lifetime of p-AuNP@HTT gradually decreases and shows a linear relationship: for every 1 °C increase in temperature, the luminescence intensity decreases by 18.76 ns. The luminescence lifetime of p-AuNP@HTT has good temperature sensitivity. The greatest advantage of using luminescence lifetime for temperature measurement is that the temperature conversion relationship is uniquely determined by the luminescence lifetime and is not affected by other external conditions such as changes in the intensity of the excitation light source, fiber optic transmission efficiency, concentration changes, etc. Therefore, it has obvious advantages over the temperature measurement method using light intensity as the temperature sensing signal.

[0132] Example 7.

[0133] Measure the pH stability of p-AuNP@HTT:

[0134] To explore the stability of the luminescence intensity of p-AuNP@HTT within the plant physiological pH range, the pH of the p-AuNP@HTT solution was adjusted to six gradients of 5.1, 6.3, 7.4, 8.1, 9.4, and 10.1 respectively with a buffer solution, and the luminescence intensity of p-AuNP@HTT under different pH conditions was measured.

[0135] Figure 12 The figure shows the schematic diagram of the stability of the luminescence intensity of p-AuNP@HTT within the plant physiological pH range. The abscissa is the pH, and the ordinate is the relative luminescence intensity (dimensionless).

[0136] As Figure 12As shown, in the range of pH 5.0 - 10.0, with the increase of pH, the relative luminescence intensity of p - AuNP@HTT is relatively stable. Therefore, the influence of the change of internal pH of plants on the luminescence intensity during temperature change can be excluded, improving the accuracy of the acquisition results.

[0137] Example 8.

[0138] Measure the Ca of p - AuNP@HTT 2+ Stability:

[0139] Ca 2+ As a second messenger, it participates in the response of plants to environmental temperature changes during temperature stress. Therefore, excluding the influence of the change of Ca 2+ ion concentration on the luminescence intensity of p - AuNP@HTT is crucial for the accuracy of the acquisition results. Measure the luminescence intensity of p - AuNP@HTT with the change of Ca 2+ concentration (10 －8 -10 －2 mol / L).

[0140] Figure 13 Shows the schematic diagram of the change of the luminescence intensity of p - AuNP@HTT with the change of Ca 2+ concentration. The abscissa is the Ca 2+ concentration mol / L, and the ordinate is the relative luminescence intensity (dimensionless).

[0141] As Figure 13 shown, in the range of plant calcium ion concentration from 10 －8 to 10 －2 mol / L, the relative luminescence intensity of p - AuNP@HTT remains stable. Thus, it can be seen that p - AuNP@HTT has very good calcium ion stability. Therefore, the influence of the change of internal ion concentration of plants on the luminescence intensity during temperature change can be excluded, improving the accuracy of the acquisition results.

[0142] Example 8.

[0143] Comparison of time - lapse imaging of HeLa cells labeled with p - AuNP@HTT and time - lapse imaging of HeLa cells labeled with the dye Hoechst.

[0144] Figure 14 Respectively show the schematic diagrams of the time - lapse imaging of HeLa cells labeled with p - AuNP@HTT and HeLa cells labeled with Hoechst dye.

[0145] After incubating Hela cells with Hoechst dye or p - AuNP@HTT respectively, fluorescence is collected after 0 ns, 50 ns, 100 ns, 300 ns, and 500 ns of time - lapse respectively. By Figure 14It can be seen that when the delay is 500 ns, a strong luminescence signal of the p-AuNP@HTT thermosensitive probe can still be observed. However, the fluorescence signal of the Hoechst dye cannot be detected after 50 ns.

[0146] The luminescence lifetime of p-AuNP@HTT can reach the microsecond level, which is not only much longer than the luminescence lifetime of autofluorescence but also much longer than the luminescence lifetime of the Hoechst dye. Therefore, p-AuNP@HTT is very suitable as a thermosensitive probe for removing the interference of autofluorescence.

[0147] Example 9.

[0148] Measure the temperature inside the tobacco leaf using the thermosensitive probe p-AuNP@HTT:

[0149] Inject 50 μl of p-AuNP@HTT with a concentration of 80 μg / ml into the tobacco leaf using a syringe. After 8 hours, put the tobacco leaf into a temperature control box. Using a PI-MAX4 (commercially available) time-gated imaging system, continuously change the environmental temperature while completely filtering out the autofluorescence background at a delay of 100 ns, and collect and record in real time the changes in the luminescence intensity of p-AuNP@HTT in the tobacco leaf and the environmental temperature. After the collection is completed, analyze the correspondence between the change in the internal temperature of the tobacco leaf and the change in the environmental temperature.

[0150] Figure 15 The schematic diagram shows the change in the luminescence intensity of the thermosensitive probe p-AuNP@HTT inside the tobacco leaf under high-temperature stress with respect to the environmental temperature. The abscissa is time in s, and the ordinate is the environmental temperature in °C and the relative luminescence intensity (dimensionless relative quantity).

[0151] Figure 16 The schematic diagram shows the correspondence between the change in the internal temperature of the tobacco leaf and the change in the environmental temperature under high-temperature stress. The abscissa is time in s, and the ordinate is the relative temperature change. The curve with floating points represents the change in the environmental temperature, and the curve without floating points represents the change in the tobacco temperature.

[0152] Figure 17 The schematic diagram shows the change in the relative luminescence intensity of the thermosensitive probe p-AuNP@HTT inside the tobacco leaf under low-temperature stress with respect to the environmental temperature. The abscissa is time in s, and the ordinate is the environmental temperature in °C and the relative luminescence intensity (dimensionless relative quantity).

[0153] Figure 18 The schematic diagram shows the correspondence between the change in the internal temperature of the tobacco leaf and the change in the environmental temperature under low-temperature stress. The abscissa is time in s, and the ordinate is the relative temperature change in °C.

[0154] As Figure 15As shown, under high-temperature stress, the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT inside tobacco changes with the ambient temperature: when the ambient temperature rises, the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT decreases, and vice versa. In the range of 0 - 400 s, the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT inside the tobacco leaf remains constant with the constant ambient temperature; in the range of 400 - 800 s, the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT inside the tobacco gradually decreases with the increase of the ambient temperature; in the range of 800 - 1200 s, the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT inside the tobacco gradually increases with the decrease of the ambient temperature. Moreover, the change amplitude of the luminescence intensity of the temperature-sensitive probe p-AuNP@HTT is synchronous with the ambient temperature, indicating its fast time response to temperature changes and can be used for the detection of rapid temperature change events / phenomena.

[0155] As Figure 16 shown, further analysis reveals that the change amplitude of the ambient temperature is higher than that of the temperature inside the tobacco leaf, reflecting that there may be a temperature self-adaptive mechanism inside the tobacco leaf to "resist" the change of the ambient temperature, which may be related to the self-protection mechanism of the tobacco.

[0156] Figure 17 and Figure 18 respectively show the change of the temperature inside the tobacco leaf with the ambient temperature under low-temperature stress. Similar to the high-temperature stress situation, there may also be a low-temperature stress adaptation mechanism inside the tobacco to "resist" the change of the ambient low temperature.

[0157] Example 10.

[0158] Use the temperature-sensitive probe p-AuNP@HTT to measure the temperature in tomatoes:

[0159] Figure 19 shows a schematic diagram of the change of the luminescence intensity inside the tomato with the ambient temperature under low-temperature stress. The abscissa is time in s, and the ordinate is the ambient temperature in °C and the relative luminescence intensity (relative fluorescence intensity) respectively.

[0160] Figure 20 shows a schematic diagram of the correspondence between the temperature change amount inside the tomato and the ambient temperature change amount under low-temperature stress. The abscissa is time in s, and the ordinate is the relative temperature change in °C.

[0161] Inject 50 μl of p-AuNP@HTT with a concentration of 80 μg / ml into the tomato leaf with a syringe. After 8 hours, put the tomato into a temperature control box. When using PI-MAX4 to completely filter the plant autofluorescence background with a delay of 100 ns, while changing the ambient temperature, collect and record the changes of the luminescence intensity of p-AuNP@HTT in the tomato leaf and the ambient temperature in real time. After the collection is completed, analyze the correspondence between the temperature change amount inside the tomato leaf and the ambient temperature change amount.

[0162] Figure 19 and Figure 20 It shows that the temperature change range in tomatoes under low - temperature stress is less than that of the ambient temperature, reflecting that there may also be a low - temperature stress adaptation mechanism in tomatoes, which can "resist" the low - temperature change of the environment.

[0163] Example 11.

[0164] Preparation of polymer - coated luminescent gold nanoparticles AuNP@DHLA (p - AuNP@DHLA) with dihydrolipoic acid as the surface ligand and measurement of the temperature sensitivity of its luminescence intensity.

[0165] First, mix 1.0 mL of a solution of PMMA - co - MAA copolymer in tetrahydrofuran (concentration: 1.0 mg / mL) and 1.0 mL of a solution of AuNP@DHLA in tetrahydrofuran (concentration: 1.0 mg / mL), and then slowly add 20 mL of ultrapure water drop - by - drop at 37 °C while continuously stirring during the dropping process.

[0166] Then, react for 10 minutes and remove tetrahydrofuran by a vacuum rotary evaporator at 45 °C to obtain a solution containing the temperature - sensitive probe p - AuNP@DHLA.

[0167] Figure 21 It shows a schematic diagram of the trend of the luminescence intensity of p - AuNP@DHLA changing with the ambient temperature. The abscissa is the temperature in °C, and the ordinate is the normalized luminescence intensity.

[0168] As Figure 21 shown, the experimental results show that after a delay of 100 ns, when the temperature rises from 20 °C to 50 °C, the luminescence intensity of p - AuNP@DHLA gradually decreases with the increase of the ambient temperature and still shows a good linear relationship: the fitting function is: y = - 0.01663x + 1.3514, and the correlation coefficient is R 2 = 0.99. When the temperature increases by 1 °C, the luminescence intensity decreases by 1.66%. Therefore, p - AuNP@DHLA is suitable for time - gated imaging to detect the internal temperature of plants.

[0169] Example 12.

[0170] Measure the internal temperature of tobacco leaves using the temperature - sensitive probe p - AuNP@DHLA:

[0171] Inject 50 μl of p-AuNP@DHLA with a concentration of 100 μg / ml into tobacco leaves using a syringe. After 8 hours, place the tobacco leaves in a temperature-controlled chamber. Using a PI-MAX4 time-gated imaging system, with the spontaneous fluorescence background completely filtered out at a delay of 100 ns, change the environmental temperature and collect and record in real time the changes in the luminescence intensity of p-AuNP@DHLA in the tobacco leaves and the environmental temperature. After the collection is completed, analyze the change in the internal temperature of the tobacco leaves with respect to the change in the environmental temperature. The experimental results show that p-AuNP@DHLA can be used as a temperature-sensitive probe to accurately detect the temperature inside tobacco leaves.

[0172] Example 13.

[0173] Preparation of polymer-clad cadmium selenide sulfide quantum dots CdSeS QDs (p-CdSeS QDs) and measurement of the temperature sensitivity of their luminescence intensity.

[0174] First, mix 1.0 mL of a solution of PMMA-co-MAA copolymer in tetrahydrofuran (concentration 1.0 mg / mL) and 1.0 mL of a solution of CdSeS quantum dots in tetrahydrofuran (concentration 1.0 mg / mL), then slowly add 20 mL of ultrapure water drop by drop at 37 °C, with continuous stirring during the dropping process. Then, react for 10 minutes. Finally, remove the tetrahydrofuran using a vacuum rotary evaporator at 45 °C to obtain a solution containing the temperature-sensitive probe p-CdSeS QDs.

[0175] The size of p-CdSeS QDs is 92.2 ± 4.8 nm.

[0176] Figure 22 The schematic diagram shows the change in the luminescence intensity of p-CdSeS QDs with the environmental temperature. The abscissa is the temperature in °C, and the ordinate is the relative luminescence intensity.

[0177] As Figure 22 shown, after a delay of 50 ns, as the temperature increases from 20 °C to 50 °C, the luminescence intensity of p-CdSeS QDs gradually decreases with the increase in the environmental temperature and still shows a good linear relationship: the fitting function is: y = -1.8x + 134.1, and the correlation coefficient is R 2 = 0.994. For every 1 °C increase in temperature, the luminescence intensity decreases by 1.8%. Therefore, p-CdSeS QDs are suitable for time-gated imaging for temperature detection.

[0178] Example 14.

[0179] Use the temperature-sensitive probe p-CdSeS QDs to measure the temperature inside tobacco leaves:

[0180] Inject 50 μl of p-CdSeS QDs with a concentration of 100 μg / ml into tobacco leaves using a syringe. After 8 hours, place the tobacco leaves in a temperature-controlled chamber. Using a PI-MAX4 (commercially available) time-gated imaging system, continuously change the ambient temperature while completely filtering out the autofluorescence background with a delay of 100 ns, and collect and record in real time the luminescence intensity of p-CdSeS QDs in the tobacco leaves and the change in ambient temperature. After the collection is completed, analyze the corresponding relationship between the change in internal temperature of the tobacco leaves and the change in ambient temperature.

[0181] Figure 23 It shows a schematic diagram of the change in internal luminescence intensity of tobacco leaves with the change in ambient temperature under high-temperature stress. The abscissa is time in s, and the ordinates are ambient temperature in °C and relative luminescence intensity (relative fluorescence intensity, dimensionless).

[0182] Figure 24 It shows a schematic diagram of the change in the amount of internal temperature change of tobacco leaves with the change in ambient temperature under high-temperature stress. The abscissa is time in s, and the ordinate is relative temperature change in °C.

[0183] Figure 23 and Figure 24 It shows that the temperature change range in tobacco under high-temperature stress is less than the ambient temperature change, reflecting that there may also be a low-temperature stress adaptation mechanism in tobacco that can "resist" environmental low-temperature changes.

[0184] Example 15.

[0185] Preparation of Eu(tta)3dpbt nanoparticles (p-EuNPs) with a polymer coating and measurement of the temperature sensitivity of their luminescence intensity:

[0186] First, mix 1.0 mL of a solution of PMMA-co-MAA copolymer in tetrahydrofuran (concentration 1.0 mg / mL) and 1.0 mL of a solution of Eu(tta)3dpb in tetrahydrofuran (concentration 0.1 mg / mL);

[0187] Then, slowly add dropwise to 20 mL of ultrapure water at 37 °C while continuously stirring during the dropping process.

[0188] Then, react for 10 minutes.

[0189] Finally, remove tetrahydrofuran at 45 °C using a vacuum rotary evaporator to obtain a solution containing the temperature-sensitive probe p-EuNPs.

[0190] The experimental results show that under the time-gated imaging system, after a delay of 500 ns, the temperature rises from 20 °C to 55 °C, and the luminescence intensity of p-EuNPs gradually decreases as the ambient temperature increases and still shows a good linear relationship: y = -1.78x + 144.32, with a correlation coefficient of R 2 = 0.994. For every 1 °C increase in temperature, the luminescence intensity decreases by 1.78%. Therefore, p-EuNPs are suitable for temperature detection in time-gated imaging.

[0191] Although the present invention has been shown and described with respect to several embodiments, it will be apparent to those skilled in the art that many changes, alterations, and alternative forms may be contemplated by those skilled in the art without departing from the spirit and scope of the invention. It is to be understood that the appended claims are intended to define the scope of the invention and thus cover equivalents or alternatives within the scope of these claims.

Claims

1. A method for detecting the internal temperature of a plant body, characterized in that, Comprising: Injecting a temperature-sensitive probe into the interior of a plant to obtain a plant containing the temperature-sensitive probe; Exciting the temperature-sensitive probe to cause the temperature-sensitive probe to emit a luminescence signal; Delayed acquisition of the luminescence signal to obtain a sample signal; Determining the internal temperature of the plant according to the sample signal; Wherein, the delay is less than the luminescence lifetime of the temperature-sensitive probe and greater than the luminescence lifetime of the plant's autofluorescence.

2. The method according to claim 1, wherein: Delayed acquisition of the luminescence signal includes: Setting initial parameters to acquire the luminescence signal and generating a real-time image; Inputting the real-time image into a discrimination model to obtain a real-time offset; Adjusting the initial parameters to make the real-time offset meet a preset threshold; The discrimination model is obtained by the following method: Previously acquiring the luminescence signal at multiple different offsets to obtain multiple reference images; Constructing a discrimination model including the correspondence between the reference images and the offsets.

3. The method according to claim 1, wherein: The luminescence signal includes at least one of luminescence intensity, luminescence lifetime, and the main peak of the luminescence spectrum.

4. The method according to claim 1, wherein: The luminescence lifetime of the temperature-sensitive probe is greater than 20 ns.

5. The method according to claim 1, wherein: The luminescence lifetime of the temperature-sensitive probe is greater than 100 ns.

6. The method according to claim 1, wherein: The delay is set to be greater than or equal to 10 ns.

7. The method according to claim 6, wherein: The delay is set to 50 ns, 100 ns, 300 ns, or 500 ns.

8. The method according to any one of claims 1 to 7, wherein: The temperature-sensitive probe is prepared by embedding a luminescent material in a polymer material; The luminescent material is selected from one or more of the following materials: semiconductor quantum dots, carbon quantum dots, rare earth luminescent materials, polymer particles containing fluorescent dyes, silica fluorescent nanoparticles, gold nanoclusters, luminescent gold nanoparticles; Wherein, The semiconductor quantum dots are selected from one or more of the following materials: CdSe, CdSe@ZnS, CdSeS, CdSeS@ZnS, CdTe, CdTe@ZnS quantum dots; The rare earth luminescent materials are selected from one or more of the following materials: rare earth complex nanoluminescent materials containing one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y); rare earth-doped upconversion nanoparticles; The fluorescent dyes are selected from one or more of the following materials: fluorescein isothiocyanate, rhodamine.

9. The method according to claim 8, wherein: The luminescent gold nanoparticles are obtained by a photoreaction, and the particle size range is 2 - 100 nm.

10. The method according to any one of claims 1 - 7, wherein: The luminescent material of the temperature-sensitive probe is selected from one or more of the following materials: NaYF4,Yb,Er@NaYF4,Yb,Nd, NaYF4,Yb,Tm@NaYF4,Yb,Nd, NaYF4:Yb,Er.

11. The method according to any one of claims 1-7, characterized in that the temperature-sensitive probe is selected from one or more of the following substances: CdS@ZnS, CdSe@ZnS, CdTe@ZnS, CdSeS, CdTeS, CdSeS@ZnS, CdTeS@ZnS quantum dots; AuNP@DHLA, AuNP@HTT, p-AuNP@DHLA, p-AuNP@HTT; Eu(tta)3dpbt, where Eu represents europium, dpbt represents 2-(N,N-diethylaniline-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine, and tta represents diacyl trifluoroacetone.

12. The method according to any one of claims 1 to 7, characterized in that the temperature-sensitive probe is prepared by embedding the luminescent material in a polymer material, including: mixing the polymer material and the luminescent material in a tetrahydrofuran solution to obtain a first mixture, dropping the first mixture into ultrapure water and continuously stirring during the dropping process to obtain a second mixture; standing the second mixture for 10-15 min to obtain a third mixture; removing tetrahydrofuran from the third mixture to obtain a solution containing the temperature-sensitive probe.

13. A temperature-sensitive probe for detecting the internal temperature of a plant body, characterized in that the luminescence lifetime of the temperature-sensitive probe is greater than 20 ns; the temperature-sensitive probe is prepared by embedding the luminescent material in a polymer material; the luminescent material is selected from one or more of the following materials: semiconductor quantum dots, carbon quantum dots, rare earth luminescent materials, polymer particles containing fluorescent dyes, silica fluorescent nanoparticles, gold nanoclusters, luminescent gold nanoparticles; wherein the semiconductor quantum dots are selected from one or more of the following materials: CdSe, CdSe@ZnS, CdSeS, CdSeS@ZnS, CdTe, CdTe@ZnS quantum dots; the rare earth luminescent materials are selected from one or more of the following materials: rare earth complex nanoluminescent materials containing one or more of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y); rare earth-doped upconversion nanoparticles; the fluorescent dyes are selected from one or more of the following materials: fluorescein isothiocyanate, rhodamine.

14. The temperature-sensitive probe according to claim 13, characterized in that the temperature-sensitive probe is selected from one or more of the following substances: CdS@ZnS, CdSe@ZnS, CdTe@ZnS, CdSeS, CdTeS, CdSeS@ZnS, CdTeS@ZnS quantum dots; AuNP@DHLA, AuNP@HTT, p-AuNP@DHLA, p-AuNP@HTT; Eu(tta)3dpbt, where dpbt represents 2-(N,N-diethylaniline-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine and tta represents dibenzoylmethane.

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