Visualization of the in situ distribution of contents and hydrogen bonding states of cellular level water in apple tissues by confocal Raman microscopy

Raman spectroscopy has been employed for studying the hydrogen bonding states of water molecules for decades, however, Raman imaging data contain thousands of spectra, making it challenging to obtain information on water with different hydrogen bonds. In the current study, a novel method combining confocal Raman microscopy (CRM) imaging with the iterative curve fitting algorithms was developed to determine the distribution of water contents at the cellular level and water states with different hydrogen bonds in apple tissues. Raman imaging data ranging from 2700 to 3800 cm(-1) were acquired from whole cells in the apple tissue, which were then decomposed into seven sub-peaks using the fixed-position Gaussian iterative curve fitting (FPGICF) algorithm. The content and hydrogen bonding states of cellular water were calculated as the area sum of the OH stretching vibration and the area ratio of DA-OH over DDAA-OH stretching vibration or the number of hydrogen bonds of each water molecule, respectively. Finally, the area of each sub-peak, the area sum of the OH stretching vibration, and the area ratio of DA-OH over DDAA-OH stretching vibration were used to visualize the distribution of each sub-peak, water contents and water states with different hydrogen bonds, respectively. In addition, it was found that the number of hydrogen bonds of each water molecule could also be considered as a criterion to describe the hydrogen bond states of water in apple tissues. The availability of such information should provide new insights for future study of cellular water in other food materials.