Note: Descriptions are shown in the official language in which they were submitted.
Dynamic Characterization Method For Micro-Nano Celluloses
Technical field
The invention relates to the field of characterization of nanocellulose
from flexible materials, in particular to characterization methods of plant-
s derived micro-nanocellulose materials.
Background art
Micro-nanocellulose has excellent properties which is
environmentally friendly, natural and recyclable, biocompatible, and has
excellent optical and mechanical properties. At present, micro-
n nanocellulose materials have been applied in various fields, such as
cosmetics, biomedicine, construction, food, military, paper, environmental
protection, etc., and have great prospects.
Current characterization methods for micro-nanocellulose include
TEM/SEM, AFM (atomic force microscopy), and DLS (dynamic light
15 scattering), etc. The existing methods are time consuming, complicated
in
preparation, and difficult to operate.
Summary of the invention
The main object of the present invention is to overcome the
shortcomings and deficiencies of the prior art, and to provide A dynamic
20 characterization method for micro-nanocellulose, which uses the existing
particle tracking velocity measuring technique to capture nanocellulose in
a microfluidic channel observed under a microscope by a CCD camera.
1
Date Recue/Date Received 2020-11-25
Then parameters such as length, diameter, speed and quantity of the
nanocellulose may be obtained by analyzing data derived from the image
captured by the CCD camera.
In order to achieve the above object, the present invention provides
the following technical solutions:
A dynamic characterization method for micro-nanocellulose
comprises the following steps:
(1) intermittently sonicating a micro-nanocellulose suspension for 5-
10min with an interval of 3s;
(2) injecting the micro-nanocellulose suspension treated by step (1) to
a micro-nano scale microfluidic channel by a mirco-injector;
(3) adjusting an objective lens of a microscope to ensure the
microfluidic channel is in within the field of view of the microscope and
exhibits a clear image, then observing how the micro-nanocellulose flows
in the microfluidic channel by the microscope to confirm that the micro-
nanocellulose is flowing in the channel at a suitable velocity;
(4) using a microscope directly connected CCD camera to capture the
micro-nanocellulose in the microfluidic channel, and keeping all positions
unchanged during the capture to reduce errors; after the capture, moving a
stage to measure the parameters of the micro-nanocellulose in the
microfluidic channel at different positions;
(5) transmitting photos taken by the CCD camera to a computer for
2
Date Recue/Date Received 2020-11-25
image data processing, and distinguishing nanocellulose from water
according to different pixel gray values of the nanocellulose and the water
on an image; calculating the length and diameter of the nanocellulose by
plotting a minimum circumscribed rectangle and an inscribed circle based
on the micro-nanocellulose profile; calculating the velocity of the
nanocellulose by center point displacement of the nanocellulose in multiple
frames; and calculating the quantity of the nanocellulose by counting the
amount of nanocellulose flowing through the system for a certain period.
Preferably, step (1) is specifically as follows:
intermittently sonicating the 0.1% micro-nanocellulose suspension
for 10 min with a mode of sonication for 3s followed by an interval of 3s
at a power of 300 Wand a temperature of 0-4 C.
Preferably, in step (3), said suitable velocity is 0-200um/s, and if the
velocity is beyond the range, the observation is not conducted until the
velocity is lowered.
Preferably, in step (4), said parameters of the micro-nanocellulose in
the microfluidic channel measured by moving a stage at different positions
comprises length, diameter, velocity and quantity of the nanocellulose.
Preferably, step (5) further comprises identification of the micro-
nanocellulose with the following process:
when the micro-nanocellulose move in the field of view, in
consecutive frames, the micro-nanocellulose move continuously along the
3
Date Recue/Date Received 2020-11-25
flow direction, while the optical properties of the suspension and the
background during the observation are stable without change, so that in a
plurality of the consecutive frames or even in the entire video, the imaging
of the background on the CCD is invariant, and moving nanocellulose in
the video is identified; wherein the micro-nanocellulose in the micro-
nanocellulose suspension are irradiated by a light source to produce weak
scattered light, and after imaging on a CCD camera, the micro-
nanocellulose has a different profile from the background with a brighter
center and darker edge.
Preferably, in step (5) length and diameter of the nanocellulose is
calculated by:
in one frame of the image, identifying the nanocellulose by the
background difference: based on the gray value difference between the
nanocellulose and the background, drawing a profile of the nanocellulose
with a gray value threshold, confirming the profile of the nanocellulose in
the frame, and drawing a minimum circumscribed rectangle on the
nanocellulose profile to enclose the micro-nanocellulose, so that the long
side of the rectangle is the length of the micro-nanocellulose, and the length
is recorded in units of pixels; and drawing an inscribed circle within the
profile of the nanocellulose, so that the diameter of the inscribed circle is
the diameter of the nanocellulose, and the diameter is recorded in units of
pixels; and
4
Date Recue/Date Received 2020-11-25
according to the size of the field of view determined by the
magnification of the microscope, and the resolution of the CCD,
determining the actual length of one pixel in the image, thereby converting
the pixel unit into a length unit.
Preferably, in step (5) velocity of the micro-nanocellulose is
calculated by:
identifying a nanocellulose, determining the center point position of
the imaging profile of the nanocellulose, and analyzing the displacement
of the center point of the nanocellulose in two consecutive frames, thereby
obtaining the displacement of the center point of the nanocellulose in
certain time interval; then calculating the velocity of the nanocellulose at
the moment by V=S/t, and in a stable flow, calculating an average velocity
of the nanocellulose by continuous analysis of multiple frames.
Preferably, in step (5) the quantity of the micro-nanocellulose is
calculated by:
given that the nanocellulose flows in from one side of the field of view
in the flowing liquid and flows out from the other side, treating the frame
where nanocellulose is identified by the system at the edge of the flow-in
side of the nanocellulose in the field of view as a first frame, identifying
and tracking each subsequent frame until the nanocellulose disappears on
the other side of the field of view, wherein the nanocellulose is counted as
1; then repeating the process to identify and track multiple targets
5
Date Recue/Date Received 2020-11-25
simultaneously so that a certain amount of nanocellulose is observed in a
certain period of time, and labeling the cellulose in the order in which the
fibers are identified by the system automatically; wherein the labeling
method is specifically: labeling the nanocellulose sequentially entered in
the field of view as 1, 2, 3, 4...n, respectively, so that starting from 1
till the
end of the video, the label of the cellulose indicated by the system is the
number of nanocellulose identified in the video.
Compared with the prior art, the present invention has the following
advantages:
1. The invention can perform real-time dynamic tracking and
measurement on micro-nanocellulose with short measuring time and large
amount of processed information. It can not only measure the length and
diameter of nanocellulose, but also velocity and position change of
nanocellulose in the flow field; the prior methods such as SEM/TEM/AFM
measure size of the solid nanocellulose statically, and the nanocellulose
needs to be dried before the measurement, therefore the existing
measurement for nanocellulose is time consuming and complicated in
operation. Compared with the existing methods, the present invention has
the advantages of short time, simple operation, and availability of multiple
parameters at the same time.
2. The present invention can count the distribution of parameters such
as number, length, and diameter of nanocellulose in the channel, which is
6
Date Recue/Date Received 2020-11-25
not possible in the prior art.
Brief description of the drawings
Fig. 1 is a flow chart of a dynamic characterization method for micro-
nanocellulose according to the present invention;
Fig. 2 shows the effect of irradiation by a light source to nanocellulose
according to the present invention;
Fig. 3 shows a circumscribed rectangle and an inscribed circle of the
profile of a nanocellulose according to the present invention;
Fig. 4 is a schematic structural view of an experimental apparatus of
1.0 Example 2;
Fig. 5 shows nanocellulose of Example 2 in a channel;
Fig. 6 is a statistical diagram of the length of nanocellulose in
Example 2;
Fig. 7 is a statistical diagram of the width of nanocellulose in Example
2;
Fig. 8 is a statistical diagram of the velocity of nanocellulose in
Example 2.
Embodiments
The present invention will be further described in detail below with
reference to the embodiments. The advantages and features of the present
invention are more readily understood by those skilled in the art, so that
the scope of the present invention is more clearly defined. It is understood
7
Date Recue/Date Received 2020-11-25
that the specific embodiments described herein are merely illustrative of
the invention and are not intended to limit the invention.
Example 1
As shown in Fig. 1, a dynamic characterization method for micro-
s nanocellulose of Example 1 comprises the following steps:
(1) intermittently sonicating a 0.1% micro-nanocellulose suspension
for 30min with a mode of sonication for 3s followed by an interval of 3s at
a power of 300 W and a temperature of 0-4 C;
(2) injecting the micro-nanocellulose suspension treated by step (1) to
1.0 a micro-nano scale microfluidic channel by a mirco-injector;
(3) adjusting an objective lens of a microscope to ensure the
microfluidic channel is in within the field of view of the microscope and
exhibits a clear image, then observing how the micro-nanocellulose flows
in the microfluidic channel by the microscope to confirm that the micro-
n nanocellulose is flowing in the channel at a suitable velocity, wherein
said
suitable velocity is 0-200um/s, and if the velocity is beyond the range, the
observation is not conducted until the velocity is lowered;
(4) using a microscope directly connected CCD camera to capture the
micro-nanocellulose in the microfluidic channel, and keeping all positions
20 unchanged during the capture to reduce errors; after the capture, moving
a
stage to measure the parameters of the micro-nanocellulose in the
microfluidic channel at different positions;
8
Date Recue/Date Received 2020-11-25
(5) transmitting photos taken by the CCD camera to a computer for
image data processing, and distinguishing nanocellulose from water
according to different pixel gray values of the nanocellulose and the water
on an image; calculating the length and diameter of the nanocellulose by
plotting a minimum circumscribed rectangle and an inscribed circle based
on the micro-nanocellulose profile; calculating the velocity of the
nanocellulose by center point displacement of the nanocellulose in multiple
frames; and calculating the quantity of the nanocellulose by counting the
amount of nanocellulose flowing through the system for a certain period.
step (5) further comprises identification of the micro-nanocellulose
with the following process:
when the micro-nanocellulose move in the field of view, in
consecutive frames, the micro-nanocellulose move continuously along the
flow direction, while the optical properties of the suspension and the
background during the observation are stable without change, so that in a
plurality of the consecutive frames or even in the entire video, the imaging
of the background on the CCD is invariant, and moving nanocellulose in
the video is identified; wherein the micro-nanocellulose in the micro-
nanocellulose suspension are irradiated by a light source to produce weak
scattered light as shown in Fig. 2, and after imaging on a CCD camera, the
micro-nanocellulose has a different profile from the background with a
brighter center and darker edge.
9
Date Recue/Date Received 2020-11-25
Length and diameter of the nanocellulose is calculated by:
in one frame of the image, identifying the nanocellulose by the
background difference: based on the gray value difference between the
nanocellulose and the background, drawing a profile of the nanocellulose
with a gray value threshold as shown in Fig. 3. For the convenience of
observation, the image is magnified for several times so that the pixel is
clear.
Then the profile of the nanocellulose in the frame is confirmed, and
as shown in Fig. 3, a minimum circumscribed rectangle and an inscribed
circle within the profile of the nanocellulose are drawn to figure out the
length and diameter of the nanocellulose.
The inscribed circle is a circle that is inscribed in the blue profile
whose diameter is equal to that of the nanocellulose. The size information
is first recorded in pixel unit. The diameter of the inscribed circle is
approximately 3 pixels (px).
The circumscribed rectangle, as shown in Fig. 3, is a minimum
rectangle around the nanocellulose and envelopes the same, such that the
long side of the rectangle equals to the length of the nanocellulose, which
is about 17 pixels ( px).
According to the size of the field of view determined by the
magnification of the microscope, and the resolution of the CCD, the actual
length of one pixel in the image may be determined, thereby converting the
Date Recue/Date Received 2020-11-25
pixel unit into a length unit.
Velocity of the micro-nanocellulose is calculated by:
identifying a nanocellulose, determining the center point position of
the imaging profile of the nanocellulose, and analyzing the displacement
of the center point of the nanocellulose in two consecutive frames, thereby
obtaining the displacement of the center point of the nanocellulose in
certain time interval; then calculating the velocity of the nanocellulose at
the moment by V=S/t, and in a stable flow, calculating an average velocity
of the nanocellulose by continuous analysis of multiple frames.
Quantity of the micro-nanocellulose is calculated by:
given that the nanocellulose flows in from one side of the field of view
in the flowing liquid and flows out from the other side, treating the frame
where nanocellulose is identified by the system at the edge of the flow-in
side of the nanocellulose in the field of view as a first frame, identifying
and tracking each subsequent frame until the nanocellulose disappears on
the other side of the field of view, wherein the nanocellulose is counted as
1; then repeating the process to identify and track multiple targets
simultaneously so that a certain amount of nanocellulose is observed in a
certain period of time, and labeling the cellulose in the order in which the
fibers are identified by the system automatically; wherein the labeling
method is specifically: labeling the nanocellulose sequentially entered in
the field of view as 1, 2, 3, 4...n, respectively, so that starting from 1
till the
11
Date Recue/Date Received 2020-11-25
end of the video, the label of the cellulose indicated by the system is the
number of nanocellulose identified in the video.
By the abovementioned steps, dynamic characterization of
nanocellulose can be done by the present invention.
Example 2
The dynamic characterization method for nanocellulose of the present
invention is carried out by the following specific experiments:
As shown in Fig.4, the instruments used in this experiment are:
Instruments Model
Work station Lenovo P410 ( Think station) work
station
Air floating isolation platform ZPT-F-M-20-12
Microscope Japan Olympus IX73 inverted microscope
DU-897U-CS0-#BV of ANDOR, Oxford,
Imaging unit
UK
First, a 0.1% NFC (cellulose nanofibril) suspension was prepared, and
then the NFC suspension was subjected to intermittent sonication for 30
min with a mode of sonication for 3s followed by an interval of 3s at a
power of 300 W and a temperature of 0-4 C, then the micro-nanocellulose
suspension treated by the sonication is injected to a micro-nano scale
microfluidic channel by a mirco-injector.
The light source of the microscope is provided by a power module
TH4-200. When the power is on, black switch below the panel is closed.
12
Date Recue/Date Received 2020-11-25
A light source power switch and an adjustment switch of the
microscope are located in front of the base, wherein the light source switch
is on the left side, and the light source adjustment switch is on the right
side. After the light source is turned on, the intensity of the light source
is
adjusted in real time according to the real situation.
The position of the stage in XY axis direction is then adjusted so that
the microfluidic channel is aligned with the objective lens during
observation.
The knob below the microscope is a focus adjustment knob, where an
outer large knob is for coarse adjustment, and an inner small knob is for
fine adjustment. The distance (focal length) between an objective lens and
the microfluidic channel is adjusted to achieve a best observation distance.
The dial switch above the microscope is a splitter switch. When it is
on the far left side, all the light is entered into an eyepiece for manual
observation. When it is in the middle, half of the light propagates to the
eyepiece and half propagates to a computer imaging system. When it is on
the far right side, then all the light propagates into the computer imaging
system, which cannot be observed at the eyepiece.
At the beginning of the experiment, a 0.1% micro-nanocellulose
suspension is intermittently sonicated for 30min with a mode of sonication
for 3s followed by an interval of 3s at a power of 300 W and a temperature
of 0-4 C. The sonicated NFC suspension is slowly injected into a
13
Date Recue/Date Received 2020-11-25
microfluidic channel. Since a high magnification objective lens is an oil
lens, it is necessary to first drop a special oil on the lens, search for the
channel under a low magnification objective lens, and move the target
image under the low magnification lens to the center of the field of view
followed by switching to the high magnification objective lens and Fine-
tuning the focus until the image under high magnification is clear as shown
in Fig. 5. When the NFC suspension flows to a portion of the channel which
can be observed by the inverted microscope, the field of view under the
microscope is recorded by the imaging unit on the computer workstation,
and calculation is continuously conducted according to the video. Since no
subsequent injection is performed, velocity of the nanocellulose in the
channel will decrease continuously due to the viscous force of the inner
wall of the channel. When the velocity in the field of view drops to about
0-200 um/s, calculation is conducted according to the video with certain
algorithm so that parameters such as quantity, length, diameter and velocity
of the nanocellulose are obtained, as shown in Fig. 6, Fig. 7, and Fig. 8.
Fig. 6 shows a length distribution of the nanocellulose in a range of 600-
2000 nm, wherein nanocellulose with a length of 600-800 nm has the
largest quantity, and that with a length of 800-1000 nm has the second
largest quantity. Fig. 7 shows a diameter distribution of the nanocellulose,
wherein the nanocellulose with a diameter of 600-650 nm has the largest
quantity. Fig. 8 shows a velocity distribution of the nanocellulose in the
14
Date Recue/Date Received 2020-11-25
range of 0-80 um/s, wherein nanocelluloses with a velocity of 20-30 um/s
has the largest quantity.
The above embodiments are preferred embodiments of the present
invention, but the embodiments of the present invention are not limited
thereto, and any other changes, modifications, substitutions, combinations,
and simplifications thereof made without departing from the spirit and
scope of the invention should all be equivalent replacements and are
included in the scope of the present invention.
15
15
Date Recue/Date Received 2020-11-25
