| Water is one of the important components in fresh fruit and vegetable cells.The distribution of content and hydrogen bonding state of water in cells has a significant influence on the phase change and migration of water during fruit and vegetable processing.Therefore,understanding the distribution of content and hydrogen bonding state of water in fruit and vegetable cells is important for studying the change mechanism of quality and microstructure during fruit and vegetable processing.However,the existing methods are unable to visualize the content and hydrogen bonding state of water in cells simultaneously.Confocal Raman microscopy（CRM）technology has been widely applied to study the distribution of certain components in cells and analyze the hydrogen bonding network of water in different systems.While,the lack of the quantitative methods of content and hydrogen bonding state of water based on Raman spectroscopy and the time-consuming and laborious processing of large amount of imaging spectra data have hindered the application of CRM in the visualization analysis of the distribution of content and hydrogen bonding state of water in fruit and vegetable cells.Therefore,the thesis aimed to develop a visualization analysis method for studying the distribution of content and hydrogen bonding state of water in fruit and vegetables at cellular scale.Firstly,a CRM detection device with precise temperature control was constructed.Under the conditions of the detection device,the Raman spectral characteristics of deionized water with tiny volume was analyzed and the quantitative method of water content in different system was determined.Then the Raman spectra of saccharide solutions with tiny volume and different concentration was analyzed.By comparing the hydrogen bonding strength of water and the number of water molecules in pure water system and solution system,a method was built to quantify the hydrogen bonding state of water in solution system.Thus,the quantification of content and hydrogen bonding state of water in solution systems based on Raman spectroscopy was solved.Then a computational code was constructed in Matlab software to implement the automatic processing and peak fitting of a large amount of imaging spectral data.The code was based on the for-loop structure and included the S-G smoothing algorithm,air PLS baseline-corrected algorithm and FPGICF algorithm,which solved the problem of spectral processing and analysis.Then the Raman imaging spectra data of apple and potato cells were acquired by the CRM detection device and processed and peak fitted automatically by the code.Based on the peak fitting results and the quantitative methods of content and hydrogen bonding state of water,a visualization method was built to analyze the distribution of content and hydrogen bonding state of water in apple and potato cells,and the differences in the water distribution of apple and potato cells was compared.Finally,the visualization analysis method was used to quantify the water content and hydrogen bonding state of water in apple and potato tissues.The quantitative results were consistent with those of LF-NMR technique and Marinchik method,which verified the reliability and accuracy of the visualization analysis method.The main research contents of this thesis are as follows:（1）A CRM detection device with precise temperature control and tiny sample volume was constructed,and the detection stability of the system were evaluated.The effect of temperature,the rough surface of sample vessel and the total volume of deionized water on the Raman spectral characteristics of deionized water with tiny volume was investigated under the conditions of the detection device.The results indicated that the detection stability of the detection system was good and the spectral acquisition parameters had no significant effect on the hydrogen bonding state of water.Under the conditions of the detection device,the rough surface of sample vessel and the total volume of deionized water had no significant effect on the hydrogen bonding strength of water but could affect the distribution of water molecules in the vessel.The hydrogen bonding state of water was enhanced with the decrease of the temperature in the range of 0～20oC,and there was a strong linear relationship between the spectral integrated intensity and the density of water in the range of 5～20oC,indicating that the integrated intensity of Raman spectra of water reflected the number of water molecules per unit volume,which was the absolute content of water in the system.Therefore,the integrated intensity of Raman spectra of water could be used to quantify the water content in different systems.It provided a theoretical basis for the quantification of water content in fruit and vegetable cells.（2）The hydrogen bonding state of water in solution system with tiny volume was quantified based on Raman spectroscopy.The Raman spectra of glucose,fructose,sucrose,and trehalose solutions with different concentrations were processed and peak fitted.According to the peak fitting results,the hydrogen bonding strength of water and the number of water molecules in solutions was analyzed and compared with those of deionized water.The results showed that the addition of saccharide molecules enhanced the hydrogen bonding strength of water in saccharide solutions.The greater the concentration,the greater the hydrogen bonding strength of water.For the saccharide solutions with same concentration,the greater the hydration capacity of the saccharide molecules and the smaller the partial molar volume,the greater the hydrogen bonding strength of water in the saccharide solutions.In addition,the amount of water molecules in saccharide solutions decreased with the increase of concentration and the change of the amount of water molecules was also related to the hydration capacity and partial molar volume of the saccharide molecule when the concentration of the solutions was same.By comparing the hydrogen bonding strength of water and the number of water molecules in the solution systems and pure water system,a method for quantifying the hydrogen bonding state of water in solution system was established;The quantitative results of the hydrogen bonding state of water based on Raman spectroscopy had a strong linear relationship with the T2 relaxation time of the solutions measured by LF-NMR,which verified the reliability of the quantitative method.The quantitative method provided a theoretical basis for the quantification of hydrogen bonding state of water in fruit and vegetable cells.（3）According to the Raman spectral characteristics of water in fruit and vegetable cells,a computational code was constructed in Matlab software to implement the automatic processing and peak fitting of a large amount of imaging spectral data.The code was based on the for-loop structure and included the S-G smoothing algorithm,air PLS baseline-corrected algorithm and FPGICF algorithm,which solved the problem that processing a large amount of imaging spectral data was laborious and time-consuming.Then the Raman imaging spectral data of apple and potato cells were acquired by the CRM detection device and processed and peak fitted automatically by the code.Based on the peak fitting results and the quantitative methods of content and hydrogen bonding state of water,a visualization method was developed to analyze the distribution of content and hydrogen bonding state of water in apple and potato cells,and the differences in the water distribution of apple and potato cells was compared.Based on the visualization results,water in the cells was classified into different types according to the distribution position of water,including vacuole water,cell wall water,water in cell junction region,water in intercellular spaces,and water in the nucleus region.The results showed that the water contents in apple or potato cells followed the order of vacuole water>water in cell junction region>cell wall water or water in cell gaps>water in the nucleus region;the hydrogen bonding state of water in apple cells followed the order of vacuole water<water in cell junction regions<water in big cell gaps<cell wall water or water in small cell gaps<water in the nucleus region;and the hydrogen bonding state of water in potato cells followed the order of big vacuole water<small vacuole water<water in cell junction regions<cell wall water<water in the nucleus region.By analyzing the morphological characteristics of the regions where vacuole water existed,it was found that there was a big vacuole with irregular shape in apple cells and there were many small vacuoles with the round or oval shape in potato cells.In apple and potato cells,the size of water existing regions had a significant influence on the hydrogen bonding state of water.（4）The reliability and accuracy of the visualization analysis method of content and hydrogen bonding state of water in apple and potato cells was verified by comparing with other methods.Firstly,the real morphological characteristics of vacuoles in apple and potato cells was analyzed by observing the microscopic images of stained apple and potato tissues and it was found that they were same with those exhibited in the distribution images of content and hydrogen bonding state of water in apple and potato cells,which verified the reliability of the visualization results.Then,the total water content,free water content and hydrogen bonding state of free water in apple and potato tissues was quantified by the visualization analysis method.The quantitative results showed that the total water content of apple tissues was 403344a.u.,the free water content of apple tissues was 84.98%,the average value of hydrogen bonding state of free water in apple tissues was 5.82;the total water content of potato tissue was 314760a.u.,the free water content of potato tissues was 75.81%,the average value of hydrogen bonding state of free water in potato tissues was 7.92.The results indicated that the total water content and free water content in apple tissues were higher than those in potato tissues,the hydrogen bonding state of free water in apple tissues were weaker than that in potato tissues.The quantitative results of CRM visualization analysis method were consistent with the results of LF-NMR measurement and Marinchik method,which further verified the reliability and accuracy of the visualization analysis method in studying the distribution of the content and hydrogen bonding state of water in fruit and vegetable cells. |
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