CN111122521A - Method for observing EZ in liquid - Google Patents

Abstract

The invention provides a method for observing EZ in liquid, which comprises the following steps: preparing microsphere liquid; adhering the bottom of an observation entity with a hydrophilic surface to a glass slide, and taking a proper amount of the microsphere liquid to contact with the hydrophilic surface of the observation entity to form an EZ sample to be observed, wherein the bottom of the EZ sample is transparent; adjusting the focus of the laser confocal microscope to a focal plane parallel to the transparent bottom surface of the EZ sample to be observed, observing at the intersection position of the focal plane and the hydrophilic surface in the EZ sample to be observed, collecting images and dynamic changes of an EZ area in the EZ sample to be observed, and processing the images. According to the invention, the EZ in the liquid is observed by adopting the laser confocal microscope, the liquid can be focused on different focal planes, a certain layer in the middle of the microsphere liquid is selected for observation, the EZ on a certain layer of the liquid and the motion trail of the microsphere can be observed without being influenced by the floating and sinking of the microsphere.

Description

Method for observing EZ in liquid

Technical Field

The invention relates to the technical field of EZ observation, in particular to a method for observing EZ in liquid.

Background

Professor Pollack, G.H., U.S. Proc.A. written The script "The Fourth Phase of Water Beyond Solid, Liquid, and Vapor" (i.e., The Fourth Phase of water: not only Solid, Liquid, and gaseous). The book has been translated into Chinese, named as "how many answers to water" to know. In the book, the authors state that water does not only have three phases, solid, liquid and gaseous, but also has a fourth phase, the "liquid crystal state". When water contacts the surface of the hydrophilic substance, a special area with the thickness of hundreds of microns is formed on the surface of the hydrophilic substance, the water in the area is in a liquid crystal state, the area is obviously different from common water in terms of pH value, electric potential, viscosity and structure, the area is very pure in a macroscopic view and can repel all impurities in boiled water, and therefore the area is called as a repelling area, namely Exclusion Zone, EZ. Microspheres (i.e., micron-sized spheres) are usually added into water to prepare microsphere water, then the microsphere water is contacted with the surface of the hydrophilic substance, and a bright area without microspheres on the surface of the hydrophilic substance can be seen under a common microscope, wherein the bright area is EZ.

Currently, EZ is observed mainly under a normal optical microscope, the added microspheres are colorless conventional microspheres, and referring to fig. 1, under a normal microscope, a slightly yellowish Nafion film and gray (or gray black) microspheres can be seen, and a gray (or bright white) strip area therebetween is EZ. Wherein, the brightness of EZ can be adjusted by adjusting the light source of the microscope. Generally, when the light intensity of a light source is weak, EZ is gray white, and the microspheres are gray black; when a light source is enhanced, EZ becomes bright, but simultaneously the color of the microspheres also becomes light and changes from gray black to gray white, so that the contrast of an EZ image observed in the prior art is low, in addition, in the test process, the conditions of microsphere sinking and adherence or floating and aggregation often occur due to the instable property of the microspheres, and in addition, the general optical microscope observes that the integral effect of all the microspheres (from top to bottom in the liquid) in the liquid is realized, and how the interaction between each microsphere and an EZ interface is not observed. Therefore, a method of more intuitively observing EZ has yet to be developed to facilitate understanding and related research into EZ.

Disclosure of Invention

In view of this, the invention provides a method for observing EZ in liquid, and aims to solve the problem that the contrast of an EZ image obtained by observation in the prior art is low.

The invention provides a method for observing EZ in liquid, which comprises the following steps: a step of preparing microsphere liquid, which is to take a proper amount of microsphere stock solution to separate microspheres, wash the separated microspheres with test liquid, and then mix the microspheres with the test liquid to obtain the microsphere liquid; the method comprises the following steps of: adhering the bottom of an observation entity with a hydrophilic surface to a glass slide, and taking a proper amount of the microsphere liquid to contact with the hydrophilic surface of the observation entity to form an EZ sample to be observed, wherein the bottom of the EZ sample is transparent; and a laser confocal microscope observation step, namely adjusting the focus of the laser confocal microscope to a focal plane parallel to the transparent bottom surface of the EZ sample to be observed, observing at the intersection position of the focal plane and the hydrophilic surface in the EZ sample to be observed, collecting images and dynamic changes of an EZ area in the EZ sample to be observed, and processing the images.

Further, in the above method for observing EZ in a liquid, the particle size of the microspheres is 0.1-10 um.

Further, in the above method for observing EZ in a liquid, the microspheres are fluorescent microspheres.

Further, in the method for observing EZ in the liquid, in the preparation step of the microsphere liquid, the microsphere stock solution is filtered to obtain microspheres, then the microspheres are filtered while being washed by the test liquid, and the microspheres on the filter membrane are washed away and mixed with the test liquid after being repeatedly washed for many times.

Further, in the above method for observing EZ in a liquid, the observation entity is a frame-shaped structure with an open top, and the bottom of the observation entity and the inner side surface with the hydrophilic surface are made of transparent materials.

Further, in the above method for observing EZ in a liquid, the observation entity is a frame-shaped structure with an opening at the top and the bottom, and the inner side surface of the frame-shaped structure with the hydrophilic surface is made of a transparent material.

Further, in the above method of observing EZ in a liquid, the observation entity is a block structure made of a hydrocolloid material.

Further, in the above method for observing EZ in a liquid, the step of confocal laser microscopy further comprises: placing an EZ sample to be observed on an adaptive clamp of a microscope maneuvering platform, starting a fluorescent grating, initially observing through an ocular lens and a 4X objective lens, and adjusting an EZ area to the middle position of a visual field; and switching the microscope objective to 10X or 20X, adjusting the focal plane to the flat and clear position of the EZ area, selecting a preset shooting mode and parameters for shooting, and recording and storing the dynamic change image of the microsphere.

Further, in the above method of observing EZ in a liquid, the step of confocal laser microscopy comprises: the laser wavelength is 400-700nm, the laser intensity is 0.01-10%, the diameter of the detection pinhole is 0.1-2AU, and the scanning speed is 0.5-4 frames/s.

According to the method for observing EZ in liquid, the EZ in the liquid is observed by adopting a laser confocal microscope, the EZ can be focused on different focal planes, a certain layer in the middle of microsphere liquid is selected for observation, the EZ on a certain layer of the liquid and the movement track of the microsphere can be observed without being influenced by the upward floating and the downward sinking of the microsphere; furthermore, fluorescent microspheres are selected as indicators, so that the observed image has more vivid color contrast.

Drawings

FIG. 1 is an EZ image observed by white microspheres and a common optical microscope in the prior art at a magnification of 100 times;

FIG. 2 is a flow chart of a method of observing EZ in a liquid provided in an embodiment of the present invention;

FIG. 3 is a schematic diagram of a structure of an observation entity according to an embodiment of the present invention;

FIG. 4 is an EZ image observed by a confocal laser scanning microscope and fluorescent microspheres at a magnification of 100 in an embodiment of the present invention.

Detailed Description

While the preferred embodiments of the present invention are described below, it should be understood that various changes and modifications can be made by one skilled in the art without departing from the principles of the invention, and such changes and modifications are also considered to be within the scope of the invention.

Referring to fig. 2, the method for observing EZ in a liquid provided by the invention comprises the following steps:

and (2) a microsphere liquid preparation step, namely separating a proper amount of microsphere stock solution to obtain microspheres, washing the microspheres obtained by separation with test liquid, and mixing the microspheres with the test liquid to obtain the microsphere liquid.

Specifically, because EZ is a repellant region that repels all impurities in the boiling water, the addition of microspheres to different types of water makes EZ observation more direct in different types of water. The particle size of the microspheres is 0.1-10um, preferably 1-10um, more preferably 5 um.

The microspheres are preferably fluorescent microspheres in order to provide a sharper image contrast in the observed EZ region. The fluorescent microspheres in this embodiment may be at least one of polystyrene fluorescent microspheres, polymethyl methacrylate microspheres, and silica microspheres.

The test liquid can be ultrapure water, distilled water, deionized water and the like, the distilled water is selected as the test liquid in the embodiment, the using amount of the test liquid can be determined according to actual conditions, in order to form the microsphere liquid in which the microspheres are suspended in the test liquid, the test liquid with the volume larger than the volume of the microsphere stock solution can be selected for washing the microspheres, the volume of the test liquid can be 3-6 times of the volume of the microsphere stock solution, and preferably, the volume of the test liquid can be 5 times of the volume of the microsphere stock solution. In this step, the concentration of the microsphere liquid can be adjusted by the test liquid, because the concentration of the microspheres is too low, so that the observed EZ edge is not clear enough due to insufficient microspheres near the EZ, and the concentration of the microspheres is too high, so that the movement track of microsphere individuals at the EZ boundary is not convenient to observe, therefore, the concentration of the microspheres is preferably 0.5-2% w/v, for example, the concentration of the microspheres can be 0.005-0.02g/ml, and more preferably 1% w/v. In this example, red fluorescent microspheres, which are purchased from Shanghai Carboxyphenanthrene biomedical science and technology Co., Ltd, with a particle size of 5um and a concentration unit of 2.5% w/v, were selected as a microsphere stock solution and treated to obtain a microsphere liquid for observation.

When the microsphere stock solution is separated, a suction filtration mode can be adopted, and the aperture of the adopted filter membrane is smaller than the particle size of the microsphere. In this embodiment, the material of the filter membrane is not limited (for example, the filter membrane may be a mixed fiber membrane), and the microspheres are conveniently washed down after the filtration.

In this embodiment, distilled water is used as a test liquid, the microsphere stock solution is subjected to suction filtration to obtain microspheres, the microspheres are subjected to suction filtration while being washed by the test liquid, and the microspheres on the filter membrane are washed away and mixed with the test liquid after being washed repeatedly. More specifically, the microspheres obtained by suction filtration are washed by distilled water for multiple times, the washing liquid can be poured off, the filter membrane is taken out of the suction filtration device, the microspheres are flushed from the filter membrane into a container by test liquid, and the microspheres and the test liquid are collected by the container, so that the microsphere liquid can be obtained.

When the method is specifically implemented, a proper amount of microsphere stock solution is taken and filtered by a filter membrane with the pore diameter smaller than the particle diameter of the microspheres, the filtered microspheres are washed by test liquid with the liquid amount which is 5 times of the volume of the microsphere stock solution, the filter membrane is washed while the filter membrane is washed for 5 times, and the microspheres on the filter membrane are washed down by the test liquid after the filter membrane is taken down, so that the microsphere liquid can be prepared.

Preparing the microspheres for observing the EZ sample: adhering the bottom of the observation entity with the hydrophilic surface to a glass slide, and taking a proper amount of the microsphere liquid to contact with the hydrophilic surface of the observation entity to form an EZ sample to be observed with a transparent bottom.

At least one of the surfaces of the observation entity which is in contact with the microsphere liquid is a hydrophilic surface, namely the inner surface and/or the outer surface of the observation entity is provided with at least one hydrophilic surface. In order to ensure the observation effect, a plurality of hydrophilic surfaces may be provided in the observation entity. In actual operation, vaseline can be used for adhering the bottom surface of the observation entity on the glass slide, and then the microsphere liquid is placed into the observation entity with the hydrophilic surface, so that the microsphere liquid is contacted with the hydrophilic surface to form an EZ sample to be observed; or the observation entity can be adhered to the glass slide by vaseline, and then the microsphere liquid is dripped on the outer surface of the observation entity, so that the microsphere liquid is contacted with the hydrophilic surface to form an EZ sample to be observed. Of course, the formation mode of the EZ sample to be observed is not limited to the above two modes, and an interface where the microsphere liquid contacts with the hydrophilic surface can be provided.

In this embodiment, the shape and size of the observation entity may be limited according to actual situations, and the shape may be regular or irregular, and the size is smaller than the size of the slide glass, in the first specific implementation manner of this embodiment, the observation entity may be in a container shape, for example: the observation entity is a frame-shaped structure with an opening at the top, and the bottom of the observation entity and the inner side surface with the hydrophilic surface are made of transparent materials. In this embodiment, the other inner side of the frame-shaped structure may also be made of a transparent material to better observe the boundary of the hydrophilic surface.

Referring to fig. 3, in a second embodiment of this embodiment, the viewing side entity may be a frame-shaped structure with an opening on the top and the bottom, and the inner side of the frame-shaped structure with the hydrophilic surface is made of a transparent material, and the slide glass can serve as the bottom of the frame-shaped structure during observation. In this embodiment, the other inner side of the frame-shaped structure may also be made of a transparent material to better observe the boundary of the hydrophilic surface.

In this embodiment, the observation entity may be made of a hydrophilic or non-hydrophilic material. In practice, when the observation entity is made of a non-hydrophilic material, a hydrophilic material may be pasted on at least one side of the observation entity contacting with the microsphere liquid to form a hydrophilic surface, and the hydrophilic material may be a Nafion membrane. When the observation entity is made of hydrophilic materials such as metal or glass, the hydrophilic surface can be formed without additionally sticking the hydrophilic materials.

In this embodiment, a plastic frame with openings at the bottom and the top printed by a 3D printer is selected as the observation entity, and the plastic frame has an outer frame with a size of 2 × 1cm, an inner frame with a size of 1 × 0.5cm, and a thickness of 0.5 cm. One bottom surface thereof was pasted on a slide glass with vaseline, a hydrophilic material (preferably Nafion film for the test phenomenon to be obvious) was cut to the size of one inner side surface (1 × 0.5 cm) of the plastic frame, and the hydrophilic material was pasted on the inner side surface of the plastic frame with vaseline. Approximately 400 ul of microsphere fluid may be placed in the 3D plastic frame prior to observation.

In a third embodiment of this embodiment, the viewing entity is a block structure made of a hydrocolloid material. Wherein, the observation entity can be made of biopolymer colloids, plant seed powder colloids, plant extract colloids, fiber and cellulose derivative colloids, starch colloids, animal hydrocolloids, pectin and/or seaweed colloids, wherein the seaweed colloids can be agar, carrageenan, alginic acid or seaweed salt; the cellulose derivative colloid can be sodium carboxymethyl cellulose; the biopolymer-like colloid can be xanthan gum or gellan gum; the plant seed powder colloid can be locust bean gum or guar gum; the animal hydrocolloid may be gelatin. The colloidal material can be mixed with a suitable solvent (the solvent can be ultrapure water, deionized water or a soluble salt solution, such as physiological saline (0.9% NaCl solution by mass)), heated for a period of time, and cooled to obtain a bulk observation entity. In the embodiment, the agar block is selected as the observation entity, and because the agar content is too low, the agar block is too soft, so that the operations in the aspects of cutting, adhering and the like before observation are inconvenient to perform; the agar content is large, the light transmittance of the resulting agar block is relatively poor, and the observation result is adversely affected, and the mass fraction of agar is preferably 1 to 3%.

In this embodiment, a square agar block with a size of 1 × 1cm is selected as the observation entity, and a plurality of drops (about 200ul to 300 ul) of microsphere liquid can be dropped around the agar block, so that the microsphere liquid is in contact with the outer surface of the agar block; of course, a fluid channel can be dug in the agar block, and the microsphere liquid is injected into the fluid channel to form a contact interface between the microsphere liquid and the hydrophilic surface.

And a laser confocal microscope observation step, namely adjusting the focus of the laser confocal microscope to a focal plane parallel to the transparent bottom surface of the EZ sample to be observed, observing at the intersection position of the focal plane and the hydrophilic surface in the EZ sample to be observed, collecting images and dynamic changes of an EZ area in the EZ sample to be observed, and processing the images.

Specifically, the confocal laser scanning microscope may be any type of microscope, and an inverted microscope is selected in this embodiment. And adjusting the focus of the laser confocal microscope to a focal plane parallel to the transparent bottom surface of the EZ sample to be observed, wherein the focal plane is preferably a parallel plane close to the transparent bottom surface. The laser confocal microscope observation step further comprises:

placing an EZ sample to be observed on an adaptive clamp of a microscope maneuvering platform, starting a fluorescent grating, initially observing through an ocular lens and a 4X objective lens, and adjusting an EZ area to the middle position of a visual field; and switching the microscope objective to 10X or 20X, adjusting the focal plane to the flat and clear position of the EZ area, selecting a preset shooting mode and parameters for shooting, and recording and storing the dynamic change image of the microsphere.

Wherein, 1024 pixels are scanned in the area, and the scanning speed is 0.5-4 frames/s, preferably 1 frame/s; the detector sensitivity HV is 1-80, preferably 44; noise reduction offset-20 to 10; preferably 0; the laser wavelength is 400-700nm, and the corresponding laser wavelength is different due to the different colors of the fluorescent microspheres, red microspheres are selected in the embodiment, and the corresponding laser wavelength is set to be 561 nm; the laser intensity may be 0.01% to 10%, preferably 2.8%, so that the brightness of the light of the microspheres remains moderate; the diameter of the detection pinhole is (0.1-2) um, and preferably 1.2 um; setting a time sequence: the interval may be longer than the imaging time of a single frame image, for example, 5 seconds.

In specific implementation, a Nikon A1HD25 laser confocal microscope laser, a control cabinet, a halogen lamp, a microscope and a computer system are started, and preheating is carried out for 10 minutes.

Placing an EZ sample to be observed on an adapting clamp of a microscope maneuvering platform, starting a fluorescence grating, starting NIS-Elements ar 5.20.00 software, selecting an eyepiece and a 4X objective lens for preliminary observation, and adjusting an EZ area to the middle position of a visual field; and switching the microscope objective to 10X or 20X, and adjusting the focal plane to a flat and clear position in the EZ area.

Selecting confocaol to shoot, scanning region 1024 pixels, scanning speed is 1 frame/S, detector sensitivity HV is 44, noise reduction offset 0, laser wavelength is 561nm, laser intensity is 2.8%, detection pinhole diameter is 1.2um, time series setting: the interval was 5 seconds. And recording the dynamic change image of the fluorescent microsphere, and finally outputting and storing in an AVI format.

Referring to fig. 4, a plastic frame printed by a 3D printing machine and having an opening at the top and bottom is used as an observation entity (a transparent inner side surface of the plastic frame is pasted with a Nafion film), under a confocal microscope, if red fluorescent microspheres are used, the reddish Nafion film and the bright red microspheres can be seen, and a black strip region therebetween is EZ. Where d represents the width of the EZ zone, measured as the observed EZ width is 100-um. It should be noted that, because the inside of the Nafion film and the light-transmitting surface face the transmission and reflection of the red light emitted by the microspheres, the inside of the Nafion film and the light-transmitting surface itself turn red, so that the boundary of the Nafion film can be clearly seen, and the region between the boundary of the Nafion film and the microspheres is the EZ region.

Furthermore, from confocal laser microscopy video recordings, it can be seen that individual microspheres exhibit random brownian motion, but have a band-shaped zone (EZ) near the hydrophilic surface into which the microspheres cannot enter, and microspheres that are flushed towards the boundary of this band-shaped zone are "bounced" back.

The microspheres are in a flowing state integrally, and after the microsphere liquid is injected into the observation entity, the liquid flows to the four sides of the frame rapidly and is rebounded by the edge of the frame, so that the microsphere liquid is in a flowing state within a period of time, and a plurality of microspheres flowing to the edge of the EZ cannot flow into the EZ.

To verify that the EZ region was observed, the present invention performed the following two tests:

firstly, observing that the width of EZ is about 100um by using the plastic frame device, then placing the device and microsphere liquid contained in the device under a white light LED lamp bead (the power is less than 0.1 w) for irradiating for 30min, wherein the lamp bead is 20cm (an LED lamp bead is adopted, and the distance of the LED lamp bead is 20cm so as to avoid the influence on the temperature of the microsphere liquid during illumination), and measuring the temperature of the microsphere liquid in the device after irradiation without changing before irradiation; after the microsphere liquid in the device is blown by a pipette three times, the device and the microsphere liquid in the device are observed under a confocal microscope, so that obvious EZ can be seen, and the width of the EZ is about 160um, which verifies that the EZ has the characteristic of becoming large (namely becoming wide) under the irradiation of visible light.

Secondly, because EZ is internally provided with negative charges, the potential in the area is obviously different from the potential of a liquid phase main body, the invention utilizes an oxidation-reduction potential microelectrode produced by Denmark unessen company to measure the potential inside and outside the EZ, the diameter of the tip of the microelectrode is 10um, and the measurement results are as follows:

position of

Nafion In the vicinity of the membrane

About from the Nafion film 25um position (EZ inner)

About from the Nafion film 50um place (EZ inner)

About from the Nafion film 100um (EZ edge) Reason)

From Nafion to 1000um (EZ) Outer)

About 2500um away from Nafion Where (outside EZ, liquid phase is dominant) Body)

Millivolt To represent Number of

-200 mV

-39 mV

-23 mV

-11 mV

-1 mV

0 mV

It can be seen from the above table that the EZ zone is negatively charged and the potential is lower than the liquid phase host.

The characteristics of the band-shaped area near the hydrophilic surface fed back in the above two experiments both conform to the characteristics of EZ (exclusionzone), i.e. it is proved that the EZ area appears between the Nafion membrane and the microsphere liquid.

In conclusion, the invention adopts the laser confocal microscope to observe the EZ in the liquid, the EZ can be focused on different focal planes, a certain layer in the middle of the microsphere liquid is selected to be observed, the EZ on a certain layer of the liquid and the motion trail of the microsphere can be observed without being influenced by the upward floating and the downward sinking of the microsphere; furthermore, fluorescent microspheres are selected as indicators, so that the observed image has more vivid color contrast.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A method of observing EZ in a liquid, comprising the steps of:

a step of preparing microsphere liquid, which is to take a proper amount of microsphere stock solution to separate microspheres, wash the separated microspheres with test liquid, and then mix the microspheres with the test liquid to obtain the microsphere liquid;

the method comprises the following steps of: adhering the bottom of an observation entity with a hydrophilic surface to a glass slide, and taking a proper amount of the microsphere liquid to contact with the hydrophilic surface of the observation entity to form an EZ sample to be observed, wherein the bottom of the EZ sample is transparent;

and a laser confocal microscope observation step, namely adjusting the focus of the laser confocal microscope to a focal plane parallel to the transparent bottom surface of the EZ sample to be observed, observing at the intersection position of the focal plane and the hydrophilic surface in the EZ sample to be observed, collecting images and dynamic changes of an EZ area in the EZ sample to be observed, and processing the images.

2. The method of claim 1, wherein the microspheres have a particle size of 0.1 to 10 um.

3. The method of claim 1, wherein the microspheres are fluorescent microspheres.

4. The method of claim 1, wherein the step of preparing the microsphere liquid comprises the steps of performing suction filtration on the microsphere stock solution to obtain microspheres, performing suction filtration while washing the microspheres with a test liquid, and washing the microspheres off the filter membrane after repeated washing for a plurality of times and mixing the microspheres with the test liquid.

5. Method of observing EZ in a liquid according to any of claims 1-4, wherein the observing entity is a frame-shaped structure with an open top, and the bottom of the observing entity and the inner side with the hydrophilic surface are made of transparent material.

6. Method of observing EZ in a liquid according to any of claims 1-4, wherein the observation entity is a frame-shaped structure open at both the top and the bottom, the inner side of the frame-shaped structure having the hydrophilic surface being made of a transparent material.

7. Method of observing EZ in a liquid according to any of the claims 1-4, wherein the observing entity is a block structure made of a hydrocolloid material.

8. The method of observing EZ in a liquid according to any one of claims 1-4, wherein the confocal laser microscopy step further comprises:

placing an EZ sample to be observed on an adaptive clamp of a microscope maneuvering platform, starting a fluorescent grating, initially observing through an ocular lens and a 4X objective lens, and adjusting an EZ area to the middle position of a visual field; and switching the microscope objective to 10X or 20X, adjusting the focal plane to the flat and clear position of the EZ area, selecting a preset shooting mode and parameters for shooting, and recording and storing the dynamic change image of the microsphere.

9. The method of claim 8, wherein the step of confocal laser microscopy comprises: the laser wavelength is 400-700nm, the laser intensity is 0.01-10%, the diameter of the detection pinhole is 0.1-2AU, and the scanning speed is 0.5-4 frames/s.

Images