Food Chemistry 385 (2022) 132679

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Visualization and quantification of content and hydrogen bonding state of

water in apple and potato cells by confocal Raman microscopy: A
comparison study
Dongmei Li a, b, c, Zhiwei Zhu a, b, c, Da-Wen Sun a, b, c, d, *
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China
b
Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
c
Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, & Guangdong Province
Engineering Laboratory for Intelligent Cold Chain Logistics Equipment for Agricultural Products, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
d
Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland,
Belfield, Dublin 4, Ireland

A R T I C L E I N F O A B S T R A C T

Keywords: Water is the most abundant component in fresh fruit and vegetables and its distribution and hydrogen bonding
Visual distribution state in cells has a significant influence on food processing. In the current study, an improved method based on
Water content our earlier studies was developed to directly visualize the spatial distribution of content and hydrogen bonding
Hydrogen bonding state of water
state of water in apple and potato cells for the first time and the difference in water distribution in these cells was
Apple and potato cells
compared. Additionally, based on the distribution images of content and hydrogen bonding state of water in
Raman imaging
Iterative curve fitting different regions in apple and potato tissues, the total water and free water contents, and the hydrogen bonding
state of free water were quantified and compared with those obtained by nuclear magnetic resonance and
Marinchik methods, demonstrating that the method could be successfully used for quantifying the content and
hydrogen bonding state of water in fruit and vegetable cells.

1. Introduction
Water is the most abundant component in fresh plant cellular food
materials, with over 80%-90% w/w in most fruit and vegetables (Khan
et al., 2016), which has a significant influence on processing, especially
on drying and freezing of fresh fruit and vegetables (Joardder et al.,
2017). Plant cellular food materials usually have complex porous tissue
structures and heterogeneous properties and water is usually located in
different compartments (such as vacuole, cytoplasm, cell wall and
extracellular space) of the cells and exhibits significant variations in the
properties and reactivity. According to the location, water in plant
cellular foods can be divided into vacuole water, cytoplasm water,
extracellular water, and cell wall water (Li et al., 2018), while according
to the self-diffusion coefficients (Dw) or bonding state, water can also be
classified as free water, loose bound water, and strong bound water.
In fruit and vegetables, it is generally accepted that water consists of
free water, loosely bound water, and strongly bound water (Caurie,
2011). However, the distribution and location of water with different
bonding states in plant cells are still vague and controversial. Cheng
et al. (2014) concluded that vacuole water was free water for its slowest
chemical exchange between water and other low molecular weight
compounds, cytoplasm and extracellular water were loosely bound
water, which was bound to the monolayer water molecules, whil cell
wall water was strongly bound water, which was bonded to the cell wall
polysaccharides, such as pectin, cellulose, and hemicellulose. However,
in the review of Joardder et al. (2017) and Khan & Karim (2017), the
intracellular water, including vacuole water and cytoplasm water, was
considered as loosely bound water and extracellular water located in the
pores and capillaries was defined as free water. Therefore, it is useful to
visualize the spatial distribution of water with different hydrogen
bonding states in plant cells for shedding more light on the issue.
In order to determine the bonding states of water in cellular food
materials, several traditional methods are used, including differential
scanning calorimetry (DSC) (Kerch et al., 2012; Peyronel & Marangoni,

* Corresponding author.
E-mail address: dawen.sun@ucd.ie (D.-W. Sun).
URL: http://www.ucd.ie/refrig, http://www.ucd.ie/sun (D.-W. Sun).

https://doi.org/10.1016/j.foodchem.2022.132679
Received 21 November 2021; Received in revised form 5 March 2022; Accepted 8 March 2022
Available online 10 March 2022
0308-8146/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Li et al.
2017), bioelectrical impedance analysis (BIA) (Cox et al., 1993), ther­
mogravimetric analysis (TGA) (Hatakeyama et al., 1988) and nuclear
magnetic resonance (NMR) (Khan and Karim, 2017). Among them, NMR
and NMR imaging (MRI) have the unique advantage in measuring the
distribution and proportion of water with different bonding states in
cellular food materials (Geng et al., 2015; Herremans et al., 2014; Kirtil
et al., 2017; Xu et al., 2017). The spin–spin (T2) relaxation in NMR is the
transverse component of the magnetization and has the potential to
investigate the cellular level water based on their intensity of relaxation,
and thus has been widely used to analyze the water distribution in the
whole sample (Khan et al., 2018; Kirtil et al., 2017), while MRI that is
based on NMR can visualize the distribution of water by spatially
gathering the intensity of relaxation of water to generate the 2D or 3D
pseudo-colour or grayscale images, which can then be used to describe
the content difference over the whole region of the sample (Westbrook &
Roth, 2011; Ajani et al., 2022). Unfortunately, the spatial resolution of
MRI micro-imaging is typically 40 µm or more, which is not sensitive
enough for characterizing the bonding states of water at the subcellular
level, and the in-situ imaging of subcellular water distribution is thus
beyond the capability of MRI. On the other hand, X-ray micro-computed
tomography technology (X-ray CT) has also been applied in the detec­
tion of water distribution and tissue microstructure of the plant cellular
materials (Almeida et al., 2016; Herremans et al., 2014; Si & Sankaran,
2016), as it can visualize the microstructure of sample tissues at a res­
olution of 1 µm, and thus can be used to acquire sufficiently high quality
images of the cells and intercellular space (Herremans et al., 2015) and
the distribution and quantity of water in the samples (Rahman et al.,
2018), however, using X-ray CT, it is not possible to distinguish the
hydrogen bonding states of cellular water in the samples.
In recent years, micro-spectroscopic techniques with a high spatial
resolution have been developed to detect components or contaminants
in agri-food products (Zhang et al., 2021a, 2021b, 2021c; Wu et al.,
2020, 2021, 2022; Hu et al., 2021; Zhou et al., 2021; Huang et al., 2021;
He et al., 2020) and to map the distribution of subcellular components
(Gierlinger et al., 2012; Horvath et al., 2012; Pan et al., 2017; Jayan
et al., 2020, 2021; Sun et al., 2021) and biological water in live cells by
using the spectral fingerprints of the molecules in the cells. Rao et al.
(2019) used fluorescence imaging microscopy to unveil the distribution
of water in the nucleolus, nucleus, cytoplasm, and lysosome in live Hela
cells based on the extent of hydrogen bonding in different regions of the
cell, while Shi et al. (2019) visualized directly the spatially-resolved
distribution of water states inside single mammalian cells by stimu­
lated Raman excited fluorescence microscopy and revealed the intra­
cellular water heterogeneity between nucleus and cytoplasm. All these
studies have proved that the micro-spectroscopic techniques have great
potential and advantage to visualize the distribution of cellular water in
live cells. However, up to now, studies on the visualization of the dis­
tribution of content and hydrogen bonding state of cellular water are
mainly focused on animal cells, few studies have been conducted on
these in plant cells.
Therefore, the authors (Li et al., 2020) successfully developed a
novel method to visualize the in-situ distribution of content and
hydrogen bonding state of water in apple cells based on the combination
of confocal Raman microscopy (CRM) imaging technology and iterative
curve fitting algorithms. In the study, the hydrogen bonding state of
cellular water was calculated by the equation proposed by Sun (2013),
but the equation is not suitable for evaluating the hydrogen bonding
state of water in plant cells for the complexity of cell saps. Subsequently,
the authors (Li et al. (2021) developed an improved equation by
comparing the characteristics of Raman spectra of saccharide aqueous
solution system and pure water system, which is given below:
Iwater
Ssolution ∝ × E0 × N (1)
Isolution
where Isolution is the integrated intensity of the Raman spectrum of
2
Food Chemistry 385 (2022) 132679
saccharide aqueous solution in the range of 3000–3800 cm− 1, Iwater is the
integrated intensity of the Raman spectrum of pure water with the same
volume as the solutions. E0 is the energy of hydrogen bonds between
water molecules in the pure water system, which is a constant at a
certain temperature. N is the average number of hydrogen bonds of each
water molecule in the aqueous solution system.
In the current study, based on the quantification method of hydrogen
bonding state of water in an aqueous solution system, the method
developed by the authors (Li et al. (2020) was further improved by
modifying the quantification method of hydrogen bonding state of
water, which was then used to analyze and visualize the distribution of
content and hydrogen bonding state of water in apple and potato cells.
Furthermore, CRM images of the distribution of cellular water in
different regions were also acquired to quantify the total water content
(TWC) and free water content (FWC), and the hydrogen bonding state of
free water in apple and potato tissues, and the results were verified by
NMR and Marinchik methods.
2. Materials and methods
2.1. Samples and reagents
Fresh apples (Red Fuji) and potatoes (NEA-303) were purchased
from a local market in Guangzhou, China and stored in a refrigerator
(BCD-535WKPZM, Midea Group, Foshan, China) at 4 ± 2 ◦ C prior to
experiments. For NMR measurements, the fresh apples and potatoes
were peeled and cut into cylinders with a diameter of 12 mm and a
height of 10 mm using a self-made steel cork-borer. For CRM and
Marinchik experiments, the prepared fresh apple or potato cylinder
samples were further cut into slices with 1 mm in thickness. The samples
used for the CRM, NMR, and Marinchik experiments were from the same
apple or potato in each trial.
D-glucose and D-fructose in the analytical grade were obtained from
Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).
Pure water was prepared by a Milli-Q system (EMD Millipore Co., Bill­
erica, MA, USA). Glucose and fructose aqueous solutions were prepared
by dissolving glucose and fructose powders into pure water at a final
concentration of 1 mol/L.
2.2. Acquisition of Raman spectra of fresh apple and potato cells
After slicing, the slices were immediately put into a closed quartz
chamber of a thermal stage (PE95/T95 System Controller, Linkam Sci­
entific Instruments Ltd., Tadworth, UK) at 20 ± 0.1 ◦ C to prevent
browning and water evaporation of the sample. For CRM experiments,
the thermal stage with the slice was placed on the sample holder of the
CRM system (LabRAM HR, Horiba France SAS, Villeneuve d’Ascq,
France) with a 10 × visible microscope objective (BX41, Olympus Co.,
Center Valley, PA, USA). In the experiments, three regions, each with an
area of about 200 × 200 µm, were selected for imaging, including a
region with an intact cell, a region with tightly connected cells, and a
region with intercellular spaces. The Raman spectra from 2700 to
3800 cm− 1 of the selected regions were acquired using the 532 nm laser
with an exposure time of 2 s and a laser power of 25% of the output
power, which was measured as 5.11 mW. The grating spectrometer was
set as 600 grooves/mm and the confocal hole was 200 µm. As cutting the
sample would damage the cell structure and water distribution on the
surface, the sample was scanned point by point at a depth of 50 µm
below the surface and the step between points was set as 4 µm. Finally,
the spectral data were converted into txt files for further processing,
which contained both spatial and spectral information of each point.
2.3. Spectral processing and visualization
The method for processing the spectra was the same as described in
Li et al. (2020). Savitzky-Golay (S-G) algorithm and adaptive iteratively
D. Li et al.
reweighted penalized least-squares (airPLS) fitting algorithm were
employed respectively to reduce noise and subtract the baseline of the
raw Raman spectra ranging from 2700 to 3800 cm− 1 for apple and po­
tato samples (Gautam et al., 2015; Zhang et al., 2010). After baseline
subtraction, a fixed-position Gaussian iterative curve fitting (FPGICF)
algorithm (O’haver, 2019) was operated as described in Li et al. (2020)
to decompose the complex and overlapping spectra into their sub-peaks
and peak parameters including area, height and width of each sub-peak,
and the fitting error and coefficient of determination were obtained. A
total of 7 sub-peaks were obtained (Li et al. (2020), including two sub-
peaks in the range of 2700–3000 cm− 1 due to CH symmetric and CH
asymmetric stretching vibrations (Gierlinger et al., 2012; Huen et al.,
2014; Pan et al., 2017; Zhang Xun et al., 2015), and five sub-peaks in the
range of 3000–3800 cm− 1, which were attributed to OH vibration of
water molecules with different hydrogen bonding motifs, including
single proton donor -double proton acceptor (DDA), double donor-
double acceptor (DDAA), single donor-single acceptor (DA), double
donor-single acceptor (DDA), and free-OH stretching vibrations (i.e., the
water molecules without hydrogen bonding) (Choe et al., 2018; Sun,
2013). Then the water content of each point was calculated as the area
sum of the five sub-peaks of the OH stretching vibration in the range of
3000–3800 cm− 1. The hydrogen bonding state of water at each point
was calculated using Eq. (1). By combining with the position coordinates
of each point, the water content and hydrogen bonding state of water
were then considered as the pixels used for generating the distribution
images of content and hydrogen bonding state of water in apple and
potato cells. The pseudo-colour distribution images were generated by
operating the self-contained pcolor and colormap functions of Matlab
2016a (MathWorks, Natick, USA) and the shading was set as interp.
2.4. Measurements of content and bonding states of water in apple and
potato tissues
NMR relaxometry experiments were performed to determine the
content and bonding states of water in apple and potato tissues. The
fresh apple and potato cylindrical samples were weighed on a digital
electronic balance (JJ1000, Changshu Shuangjie Testing Instrument
Factory, Changshu, China) with an accuracy of 0.01 g. The samples were
then put into glass tubes with a diameter of 15 mm and sealed with
parafilm and placed in a specific location of the sample chamber in a
low-field nuclear magnetic resonance imaging analyzer (LF-NMR)
(NMI20-040H-I, Suzhou Niumag Analytical Instrument Co., Ltd., Suz­
hou, China) with a spectrometer frequency of 20 MHz. The temperature
of the sample chamber was kept at 32.00 ± 0.01 ◦ C. Carre-Purcell-
Meiboome-Gill (CPMG) sequence was employed to measure spin–spin
relaxation time T2 of water in the samples. In order to obtain the time
and proportion of different T2 components, the T2 relaxation time data
were processed by simultaneous iterative reconstruction technique
(SIRT) inversion algorithm (Geng et al., 2015).
2.5. Measurements of total water and free water contents
The TWC and FWC of apple and potato tissues were measured ac­
cording to the Marinchik method described in Zhu et al. (2021). For
measuring TWC, samples were dried in a forced-air oven (DHG-9240A,
Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) at
85 ◦ C until constant weight. For determining FWC, samples were placed
into weighting bottles and mixed with 5 mL 60% (w/w) sucrose aqueous
solution, the weighting bottles were then put on a thermostatic oscillator
(SHA-C, Changzhou Aohua Instrument Ltd., Changzhou, China) at
20 ± 2 ◦ C for 5.5 h, and the final concentration of sucrose aqueous so­
lution was measured by an Abbe refractometer (WAY-2 W, Shanghai
Precious Science Instrument Co., Ltd., Shanghai, China). The TWC and
the FWC were calculated by the equations below:
3
Food Chemistry 385 (2022) 132679
W1 − W2
TWC % = × 100 (2)
W1
G × (D1 − D2 ) / D2
FWC % = × 100 (3)
W1 ⋅TWC
where W1 is the fresh weight (g) of the sample, W2 is the dry weight (g)
of the sample, G is the mass (g) of the sucrose aqueous solution, D1 is the
initial concentration of the sucrose aqueous solution and D2 is the final
concentration of the sucrose aqueous solution.
2.6. Statistical analysis
For each apple or potato, four slices were used to carry out the CRM
imaging experiments for quantifying the TWC, FWC, and hydrogen
bonding state of free water in the tissues and the average was reported.
For NMR and Marinchik experiments, all measurements were performed
in triplicate and their average was reported. All data were processed by
Microsoft Excel 2016 and Matlab R2016a (Mathworks, Natick, USA) and
expressed as means ± SD.
3. Results and discussion
3.1. Characterization of Raman spectra of apple and potato cells
The optical microscopic images of apple and potato tissues and the
raw Raman spectra acquired from apple and potato cells are shown in
Fig. 1. The red box in Fig. 1a is the imaging region in the tissues, which
contains an intact apple or potato cell. The size of the imaging region
was 220 × 240 µm for the apple cell and 200 × 220 µm for the potato
cell, and 3416 and 2856 spectral bands were acquired from the imaging
region, respectively (Fig. 1b). It is obvious that the spectra were quite
different, especially for the apple cell, which might be resulted from the
complex porous tissue structure and heterogeneous properties of plant
cells. Compared with apple tissues, the cell arrangement of potato tis­
sues was more regular and tighter, and the cell sizes and shapes were
more uniform as shown in Fig. S1. Therefore, the difference in the
spectra of the potato cells was smaller than that of apple cells.
In order to further analyze the difference, four spectra acquired from
different points in the imaging region are shown in Fig. 1c-f. The spectral
acquisition points are marked in different colours in Fig. 1a. Among
them, spectrum c and spectrum d were acquired from the red and yellow
points inside the cell, respectively, spectrum e was obtained from the
white point at the cell wall or cell junction, and spectrum f was from the
green point in the intercellular space. It could be found that the shapes of
these spectra were similar, containing two overlapping Raman peaks in
2700–3000 cm− 1 and 3000–3800 cm− 1. However, the shapes of the
spectra in 2700–3000 cm− 1 from apple and potato cells were different,
particularly, the shapes of the spectra from apple cells were similar to
those of 1 mol/L fructose aqueous solution, while the shapes of the
spectra from potato cells were similar to those of 1 mol/L glucose
aqueous solution as shown in Fig. S2. Previous studies indicated that
Raman peaks in 2700–3000 cm− 1 were mainly attributed to the C–H
stretching vibrations of carbohydrate, protein, and other biomolecules
(Choe et al., 2018; Li et al., 2020), therefore, it was reasonable to suggest
that the fructose in the apple cell sap was more than other soluble sac­
charides, while in the potato cell sap, there was more glucose. This was
consistent with other published conclusions that fructose was the most
abundant soluble sugar in apple tissues (Liu et al., 2016) and glucose
was one of the main monosaccharides in potato tissues (Cai et al., 2018).
The shapes of the Raman peaks of the spectra in 3000–3800 cm− 1
were similar to those of water. In addition, the signal to noise ratio (SNR)
of the spectra in 3000–3800 cm− 1 acquired from freeze-dried apple and
potato samples were 1.818 and 1.981, respectively, while the SNR of
spectra in this range from fresh samples were 26.516 and 26.689,
D. Li et al. Food Chemistry 385 (2022) 132679

Fig. 1. Optical microscopic image (a) of apple tissue (left) and potato tissue (right) and all raw Raman spectra (b) in the range of 2700–3800 cm− 1 of the apple and
potato cell obtained by CRM. The red box in the microscopic image is the imaging region with an intact apple or potato cell, the size of the region is 220 × 240 µm
(apple cell) and 200 × 220 µm (potato cell), respectively. The four points are the acquisition points of the spectrum c (red), d (yellow), e (white), and f (green),
respectively.

respectively (Fig. S3). Therefore, the Raman peaks of the spectra in
3000–3800 cm− 1 were assigned to the OH stretching vibration of water
molecules.
For the four spectra from the apple cells, the baselines of the spectra
from the cell wall or cell junction and intercellular space were much
higher but the spectral intensity of the Raman peak of OH stretching
vibration was lower than that of the spectra from inside the cell, indi­
cating that the water contents in the cell wall and intercellular space
were less than that inside the cell, but the contents of other cellular
components were higher, such as cellulose, hemicellulose, protein, air,
and so on, which could generate a high background signal during
spectral acquisition (Pan et al., 2017; Szymańska-Chargot et al., 2016).
For the four spectra from the potato cell, there was no obvious dif­
ference in the baselines, indicating that the distribution of cellular
components in potato cells was more homogeneous than that in apple
tissues. However, the spectral intensity of the OH stretching vibration of

Fig. 2. Preprocessed Raman spectra of the apple (a) and potato (b) cells based on S-G and airPLS algorithms; peak fitting results (c), residual plot (d) and histograms
of the fitting determination coefficients (R2) (e) of the Raman spectra of the apple and potato cells.

4
D. Li et al. Food Chemistry 385 (2022) 132679

the spectra inside the cell was higher than that in the cell wall or cell
junction and intercellular space, furtherly indicating that the content of
intracellular water was higher than that of intercellular water or cell
wall water.
3.2. Characteristics of water content distribution in cells
In order to further analyze the Raman spectra and compare the dis­
tribution of water in apple and potato cells, all the Raman spectra of
apple and potato cells were preprocessed by smoothing and baseline
subtraction, which were then decomposed into 7 sub-peaks by
combining the S-G, airPLS, and FPGICF algorithms using the round-
robin algorithm in the Matlab operating environment. Fig. 2a and
Fig. 2b show the preprocessed Raman spectra, which were smooth with
flat baselines, and the two overlapping characteristic peaks were effec­
tively retained. Fig. 2c and Fig. 2d show the results of peak fitting and
the corresponding residual plots, while Fig. 2e shows the histograms of
the fitting determination coefficients (R2). For the spectra of the apple

Fig. 3. CRM images of the distribution of content and hydrogen bonding state of water in apple and potato cells. a and b: optical microscopic images of apple and
potato tissues, respectively; c and e: Raman images of the distribution of water content in apple and potato cells, respectively; d and f: Raman images of the dis­
tribution of hydrogen bonding states of water in apple and potato cell, respectively; BCG: big intercellular space; SCG: small intercellular space; CJ: cell junction; CW:
cell wall; V: vacuole.

5
D. Li et al.
cells, R2 were greater than 0.99 with 93.91% greater than 0.999, and for
the potato cells, R2 were greater than 0.999, indicating that the spectral
processing methods used were suitable to preprocess and peak-fit a large
number of spectra.
Fig. 3a and Fig. 3b show the optical microscopic images of an intact
apple and potato cell. Based on the peak fitting results, the corre­
sponding distribution images of water contents in the cells were
generated and are shown in Fig. 3c and Fig. 3d, indicating the existence
of distinct cell borders, which was the same as that in the microscopic
images. This was because the water content of the cell wall or cell
junction was lower than that inside the cell, thus the colour of these
regions in the generated images is different from that inside the cell.
From Fig. 3c and Fig. 3d, it could also be observed that there were
several bubble-like regions with the highest water content (marked as
V), which were the vacuole regions of the cells. For the apple cell, there
was no obvious demarcation between these bubble-like regions, and it
was thus inferred that there was only a big central vacuole with an
irregular shape in the apple cell. However, for the potato cell, multiple
oval vacuoles with different sizes and regular shapes were relatively
evenly distributed inside the cell, and the bigger the size of the vacuole,
the higher the water content in the vacuole.
The regions marked with CW or CJ in Fig. 3c and Fig. 3d are located
in the cell borders, thus were assigned to the cell wall or cell junction
regions. The water contents in these regions were not homogeneous,
with higher water contents in the middle than on both sides. This was
probably related to the compositions of the cell wall, which has three
layers. Among them, the primary and secondary cell walls are mainly
composed of cellulose microfibril, hemicellulose and some soluble pro­
tein, and the middle lamella layer is the outermost layer, which forms
the interface between adjacent plant cells and links them together (Kang
et al., 2021). As the hydration capability of pectin is much stronger than
that of cellulose and hemicellulose (Paudel et al., 2016), the middle
lamella layer can bond more water than the other two layers of the cell
wall. Therefore, it could be suggested that the water content in the cell
junction region was higher than that in the cell wall.
In apple tissues, the cells were in irregular shapes and were loosely
arranged, there were thus some intercellular spaces between the cells,
which are marked with CG in Fig. 3a and Fig. 3c, indicating that the
water content in the intercellular spaces was similar to that in the cell
wall. The reason for the similarity was because most of the intercellular
spaces were generated by the degeneration or shrinkage of the middle
lamella of the cell wall in adjacent cells, leading to a similar structural
composition between the intercellular space and the cell wall.
3.3. Characteristics of distribution of hydrogen bonding state of water in
cells
The hydrogen bonding state of water at each point in the apple and
potato cells was calculated using Eq. (1) with some modifications. Iwater
in the equation could not be determined as it was impossible to acquire
the Raman spectra of pure water with the same volume and environment
of the cell sap in apple and potato cells. As the maximum integrated
intensity of the spectra in the range of 3000–3800 cm− 1 for apple and
potato cells was 8.4 × 105 and 7.2 × 105, respectively, for simplifying
the calculation, Iwater was set as 1 × 106 since the integrated intensity of
Raman spectra of pure water should be greater than that of the cell sap
under the same condition (Li et al., 2021). Therefore, Eq. (1) was
modified as.
1 × 106 IDAA × 3 + IDDAA × 4 + IDA × 2 + IDDA × 3
Ssolution ∝ × (4)
IO∙H IO∙H
where IO∙H represents the integrated intensity of the Raman spectra in
the range of 3000–3800 cm− 1 at each point in apple and potato
cells.IDAA ,IDDAA ,IDA , and IDDA represent the integrated intensity of the
sub-peaks corresponding to DAA-OH, DDAA-OH, DA-OH, and DDA-OH
6
Food Chemistry 385 (2022) 132679
hydrogen bonding motifs, respectively. Compared with Eq. (1), Eq. (4)
was more accurate for evaluating the hydrogen bonding states of water
in the cell sap, especially the regions with high water contents, such as
the vacuole regions in apple and potato cells.
Fig. 3e and Fig. 3f show the distribution of the hydrogen bonding
states of water in apple and potato cells, and the smaller the value, the
weaker the hydrogen bonding state of water. It was observed that the
hydrogen bonding states of water in the vacuole regions were the
weakest either in apple or in potato cells. For the apple cell, the
hydrogen bonding states of water in vacuole regions were relatively
uniform and the values were within the range of 4 ~ 8. However, for the
potato cell, the hydrogen bonding states of water were related to the size
of the vacuole region, and the values ranged from 4 to 7 for the big
vacuoles and from 7 to 10 for the small ones, indicating that the size of
the vacuoles could affect the hydrogen bonding states of the vacuole
water. This result was in good agreement with the conclusion of Michel
et al. (1989), who concluded that the size of water domains had a sig­
nificant influence on the strength of hydrogen bonds between water
molecules, and the bigger the size of water domains, the weaker the
hydrogen bonding strength of water. A similar result was also found in
the hydrogen bonding states of water in intercellular spaces in apple
tissues. The hydrogen bonding states of water in the bigger intercellular
spaces (marked with BCG in Fig. 3a and Fig. 3e) were weaker than those
in the smaller ones (marked with SCG in Fig. 3a and Fig. 3e), which
further confirmed that the hydrogen bonding states were strongly
affected by the size of the regions where water existed.
3.4. Quantification of TWC, FWC and hydrogen bonding state of free
water in tissues
CRM imaging method was used to quantify the TWC, FWC and
hydrogen bonding state of free water in tissues. By observing the CRM
images, the apple and potato tissues consisted of three types of regions,
including those with tightly connected cells (Fig. 4a and Fig. 4d), with
intercellular spaces (Fig. 4 b and Fig. 4e), and with an intact cell (Fig. 4c
and Fig. 4f). Therefore, in order to quantify TWC, FWC, and the
hydrogen bonding state of free water in apple and potato tissues, the
Raman spectra of these regions were acquired from each apple or potato
slice, then the corresponding distribution images of water contents and
hydrogen bonding states in these regions were generated and are shown
in Fig. 4a-f. The water content of each region was calculated as the
average water content at all pixels in the region, and the water content of
each slice was calculated as the average water content of the three re­
gions, and TWC of apple or potato tissues was defined as the average
water content of four pieces of apple or potato slices.
Image segmentation was performed on the distribution images of
water content and hydrogen bonding state of water and results indicated
that for apple tissues, most of the pixels were located in the vacuole
regions when their integrated intensity of the Raman spectra was greater
than 3.5 × 105 and the hydrogen bonding state of water was lower than
8, therefore, water at these points was defined as free water, while for
potato tissues, the integrated intensity of the Raman spectra of free
water was greater than 3.5 × 105 and the hydrogen bonding state was
lower than 10. Then the FWC of each region was calculated as by the
equation below:
IFW
FWC(%) = × 100 (5)
ITW
where, IFW is the sum of the integrated intensity of the Raman spectra
corresponding to free water in the region, ITW is the sum of the inte­
grated intensity of all Raman imaging spectra of the region.
Table 1 shows TWC, FWC, and the hydrogen bonding state of free
water in apple and potato tissues, revealing that both TWC and FWC for
apple tissues were higher than those for potato tissues, while the
hydrogen bonding state in apple tissues was weaker than that in potato
D. Li et al. Food Chemistry 385 (2022) 132679

Fig. 4. CRM images of the distribution of content (middle) and hydrogen bonding state (bottom) of water (bottom) in different regions in the apple (a, b, c) and
potato (d, e, f) tissues. The red box (upper) is the imaging region in the apple or potato tissues. a and d: the region with tightly connected cells; b and c: the region
with intercellular spaces; c and f: the region with an intact cell.

the TWC and FWC of apple tissues were higher than those of potato
Table 1
tissues, further demonstrating the reliability of the results obtained by
Average integrated intensity or TWC, FWC and hydrogen bonding state of free
the CRM imaging method.
water in apple and potato tissues acquired by CRM imaging, LF-NMR, and
Although the conclusions obtained by the three methods were
Marinchik methods.
consistent with each other, there were still some differences in the re­
Methods Samples Average integrated FWC (%) Hydrogen
sults. The ratio of the TWC of apple and potato tissues measured by the
intensity (a.u.) or bonding state of
TWC (%) free water CRM imaging method was 1.28, while those by LF-NMR and Marinchik
method were 1.06 and 1.07, respectively. This might be due to the
CRM Apple 403344 ± 89686 84.98 ± 9.83 5.82 ± 1.00
imaging tissue
limitation of CRM in measuring strongly bound water, which is usually
Potato 314760 ± 51064 75.81 ± 8.17 7.92 ± 0.37 located in the regions with low water content, and the Raman spectra of
tissue water in these regions are easily affected by noises, leading to the low
LF-NMR Apple 7132.60 ± 362.30 90.36 ± 0.91 1150.67 ± 45.87 SNR and difficulty in distinguishing them (Li et al., 2020). Meanwhile,
tissue
the content of strongly bound water in potato tissues was much higher
Potato 6710.50 ± 112.10 85.15 ± 2.06 432.76 ± 85.13
tissue than that in apple tissues as shown in the inset table in Fig. 5, therefore,
Marinchik Apple 88.91 ± 2.00 86.53 ± 2.98 – the TWC of potato tissues measured by the CRM imaging method was
tissue lower than the actual TWC of potato tissues. Additionally, the FWCs of
Potato 83.35 ± 1.24 76.66 ± 1.91 – apple and potato tissues measured by the LF-NMR method were higher
tissue
than those obtained by the CRM imaging method, which was resulted
from the higher temperature of the NMR experiments as high temper­
tissues. In addition, the standard deviation of the results for apple tissues ature could weaken the hydrogen bonding strength of water molecules
was greater than that of potato tissues, indicating that the regional (Sun, 2010). However, the FWC measured by the CRM imaging method
difference for apple tissues was bigger than that for potato tissues, was approximate to that of the Marinchik method, which verified the
further confirming that water distribution in potato tissues was more reliability and accuracy of the CRM imaging method in quantifying FWC
uniform than that in apple tissues. in apple and potato tissues.

3.5. Comparison of water content and hydrogen bonding state of water by
CRM imaging with LF-NMR and Marinchik methods
The T2 relaxation time spectra of water in apple and potato tissues is
shown in Fig. 5 and the T2 relaxation times and proportions of the area
corresponding to each peak of the spectra of water in apple and potato
tissues are listed in the inset table. According to the principle of nuclear
magnetic resonance, the smaller the relaxation time of water, the lower
the degree of freedom of protons and the stronger the hydrogen bonding
state of water. Therefore, based on the classification of water in plant
tissues, the three peaks (a-c) in Fig. 5 were corresponded to strongly
bound water, loosely bound water, and free water, respectively. By
comparing the results in Table 1, it was found that both TWC and FWC
for apple tissues were higher than those for potato tissues and the
hydrogen bonding state of free water in apple tissues was weaker than
that in potato tissues, which was consistent with the results obtained
previously by the CRM imaging method. In addition, the TWC and FWC
measured by the Marinchik method shown in Table 1 also indicated that
4. Conclusions
In this work, an improved novel method was developed based on our
earlier studies to visualize and compare the distribution of content and
hydrogen bonding state of water in apple and potato cells. The distri­
bution images of content and hydrogen bonding state of water showed
that the water contents in apple or potato cells followed the order of
vacuole water > water in cell junction regions > water in the cell wall or
intercellular spaces, and the hydrogen bonding states of water followed
the opposite order. The size of water existing regions had a significant
influence on the hydrogen bonding states of water. Results of CRM im­
aging revealed that both TWC and FWC for apple tissues were higher
than those for potato tissues and the hydrogen bonding state of free
water in apple tissues was weaker than that in potato tissues, which were
consistent with the results obtained by LF-NMR and the Marinchik
method. Therefore, this improved method was accurate and reliable in
investigating the distribution of water content and water states in apple
and potato cells and could thus be used as a new technique for

7
D. Li et al.
Fig. 5. T2 relaxation time distribution acquired from fresh apple and potato tissues by NMR relaxation experiments. The inset table shows the T2 relaxation time and
the proportion of the area of each relaxation peak of apple and potato tissues.
investigating water distribution in other plant cells. It would be bene­
ficial to explore the mechanism of water migration at the cellular level
during fruit and vegetable processing, especially freezing and drying,
which could further control and optimize the quality of the fruit and
vegetable products.
CRediT authorship contribution statement
Dongmei Li: Writing – original draft, Formal analysis, Investigation.
Zhiwei Zhu: Validation, Funding acquisition, Resources. Da-Wen Sun:
Supervision, Funding acquisition, Resources, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The authors are grateful to the Guangzhou Key Laboratory for
Intelligent Sensing and Quality Control of Agricultural Products
(202102100009) for its support. This research was also supported by the
Contemporary International Collaborative Research Centre of Guang­
dong Province on Food Innovative Processing and Intelligent Control
(2019A050519001) and the Common Technical Innovation Team of
Guangdong Province on Preservation and Logistics of Agricultural
Products (2021KJ145).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.132679.
8
Food Chemistry 385 (2022) 132679
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