Bionic blood vessel microchannel for magnetic targeted delivery research and application of bionic blood vessel microchannel

Abstract

The invention belongs to the technical field of magnetic targeted delivery, and particularly relates to a bionic blood vessel microchannel for magnetic targeted delivery research and application of the bionic blood vessel microchannel. The plant leaf chloroplast is a product obtained by replacing chloroplast and cell sap in the plant leaf with deionized water. The application comprises the following steps: pouring a magnetic microbead solution into the bionic blood vessel microchannel, guiding the movement of the magnetic microbead by guiding the movement of the magnet, and observing the movement process. According to the technical scheme provided by the invention, the micro-channel structure is prepared based on the plant veins, an unstructured cross-scale (from centimeter to micrometer) multi-stage multi-branch pipeline type structure is formed, and the structure has good transparency, so that the morphology and position distribution of the controlled magnetic particles can be observed by using a common optical lens in the driving of the magnetic micro-particles; the method has the advantages of low cost and simple process.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic targeted delivery, and in particular relates to a bionic blood vessel microchannel used for magnetic targeted delivery research and application thereof. Background Art

[0002] Magnetic targeted drug delivery technology can be divided into two types: continuous magnetic positioning and instantaneous magnetic positioning. Continuous magnetic positioning means that during the treatment process, the movement direction of magnetic nanoparticles is continuously controlled by an external magnetic field so that they move toward the target cells or organs. One of the delivery routes is the delivery of drug-loaded nanomagnetic particles through blood vessels. Therefore, achieving controllable movement of magnetic microparticles along the micro-pipeline environment under the guidance of an external magnetic field is a common and basic technology for achieving magnetic targeted delivery.

[0003] In order to better study the controllable movement of magnetic particles in blood vessels and ultimately achieve faster delivery in blood vessels, it is necessary to construct a microchannel structure that simulates blood vessels. At present, the methods for constructing microchannel structures are mainly divided into microfluidic chips made by hot pressing, molding, injection molding, laser ablation, etc., as well as various infusion tubes and spliced ​​pipes made by simple glass tubes. The microfluidic structures prepared by these methods have the obvious characteristics of structured pipeline morphology, but have the disadvantages of simple morphology, little change in tube diameter, and simple network level. In other words, there is a huge gap between its morphology and that of blood vessels, resulting in a large deviation between the simulation results and the actual situation.

[0004] In addition, in order to achieve the controllable movement of magnetic microspheres, it is necessary to be able to observe the position of the magnetic microspheres, so the prepared channel needs to have transparent properties (visible light can easily pass through) to achieve the observation and positioning of magnetic beads under a common optical microscope, thereby providing the prerequisite for the study of the controllable movement of magnetic microspheres. However, the current microchannel structure is often not possible due to material limitations, or the transparency is not very ideal, which also limits the progress of the research process. Summary of the invention

[0005] The present invention provides a bionic blood vessel microchannel for magnetic targeted delivery research, which is used to solve the problems of simple morphology, little change in tube diameter and simple network level of the current microfluidic chip.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: a bionic vascular microchannel for magnetic targeted delivery research, which refers to a product obtained by replacing the chloroplasts and cell fluid in the plant leaves with deionized water.

[0007] Optionally, the plant leaves are selected from water spinach leaves, spinach leaves, chicken feather cabbage leaves or sweet potato leaves.

[0008] Optionally, the bionic vascular microchannel for magnetic targeted delivery research is prepared by the following method, which specifically includes the following steps:

[0009] S11: Select a suitable leaf and remove the cuticle of the leaf using an exfoliating mixed solution;

[0010] S12: Soak the leaves treated by S11 in a deproteinization solution for 100-140 hours, then wash and proceed to the next step;

[0011] S13: Soak the leaves treated with S12 in the prepared cell lysis mixed solution for 10-14 hours, then wash and proceed to the next step;

[0012] S14: Infuse sterile deionized water from the petiole of the leaf blade, with the injection pressure controlled at 120-180 mmHg, until water precipitates from the leaf surface;

[0013] S15: Soak the leaves treated with S14 in sterile deionized water at 3-5°C for 10-20 days and then take them out for testing.

[0014] Optionally, the exfoliating mixed solution is a mixed solution of n-hexane aqueous solution and PBS; the deproteinizing solution is an SDS (sodium dodecyl sulfate) aqueous solution; and the cell lysis mixed solution is a mixed solution of Triton X-100 and a deionized aqueous solution of sodium chloride.

[0015] Optionally, in step S15, the temperature of the sterile deionized water is 4° C., and the duration is preferably 13-15 days.

[0016] Optionally, the detection method in step S15 is: first hang and drain until there is no water on the leaf surface, then use Ponceau aqueous solution to perfuse from the petiole to observe the integrity of the leaf and the patency of the veins.

[0017] The present invention also provides a control method for driving magnetic microspheres to move along a preset path in the bionic vascular microchannel for magnetic targeted delivery research using a magnetic field, which specifically comprises the following steps:

[0018] S21: Use a microfluidic pump to infuse 8-12 ml of a 0.1-1% (g / 100 ml) magnetic microbead solution from the petiole of the leaf blade, and place the sample into which the magnetic microbeads have been introduced on a transparent stage;

[0019] S22: According to the motion state model of the magnetic microbeads when being pulled by magnetic force in space, the adjustment data of the center position of the guiding magnet is obtained, thereby realizing the directional motion of the magnetic microbeads and observing the motion process of the magnetic microbeads using an optical microscope.

[0020] Optionally, the magnetic microbeads are micron-sized microbeads of ferroferric oxide nanoparticles coated with agarose or micron-sized immunomagnetic beads whose surfaces are modified with hydroxyl groups or fluorescent lamps.

[0021] Optionally, the concentration of the magnetic microbead solution is preferably 0.8-1% (g / 100 ml).

[0022] Optionally, when the magnetic microbeads are pulled by magnetic force in space, the motion state model in the XZ plane is as follows:

[0023]

[0024] in, It refers to the magnetic field force exerted on the magnetic beads in the X direction. It refers to the magnetic field force on the magnetic beads in the Z direction, m B refers to the mass of the magnetic microbeads, 6πηr refers to the fluid resistance of the magnetic microbeads, It refers to the speed of the magnetic beads in the X direction. refers to the speed of the magnetic microbeads in the Z direction, w refers to the X component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead, and h refers to the Z component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead;

[0025] For any w, there exists a corresponding where h balance It refers to the distance component in the Z direction between the magnetic beads and the guide magnet when the beads are in balance in the Z direction. It refers to the actual position height of the guiding magnet at this time, so that the magnetic beads maintain a force balance state in the Z direction. At this time, the magnetic beads receive the X-direction force of the permanent magnet and move at a variable speed on the X axis;

[0026] For any w, there exists a corresponding in It refers to the height position of the guiding magnet, which is a function of w, causing the magnetic beads to move at a variable speed in the Z-axis direction.

[0027] Optionally, the guiding magnet is formed by a permanent magnet, an electromagnetic coil or an electromagnetic coil group. When the guiding magnet is a permanent magnet, its center position is the geometric center; when the guiding magnet is an electromagnetic coil or an electromagnetic coil group, its center position is the center of the coil or the coil group.

[0028] Optionally, when performing step S22, the leaf vein channel needs to be kept moist, the sample is placed in a moistened surface dish and water is added regularly or a cover glass is covered on the surface of the sample.

[0029] The technical solution provided by the present invention is to prepare a microchannel structure based on plant leaf veins to form an unstructured cross-scale (from centimeters to micrometers) multi-level multi-branch pipeline structure with good transparency, which is convenient for using ordinary optical lenses to observe the morphology and position distribution of the controlled magnetic particles during the driving of magnetic microparticles, and has the advantages of low cost and simple process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 are photos of leaves at various stages of the treatment process in Example 1, a is the original leaf, b is after exfoliation, c is after protein removal, and d is after cell lysis;

[0031] Figure 2 is a schematic diagram of the hanging drainage method described in Example 1;

[0032] Figure 3 This is a photo of the perfusion of the Ponceau solution described in Example 1;

[0033] Figure 4 It is to prepare the morphological photos of the samples under the microscope after freezing section;

[0034] Figure 5 This is a photo of the implementation method of keeping the leaf vein channel moist in Example 2;

[0035] Figure 6 is a schematic diagram of the motion control of magnetic microspheres achieved by the gradient magnetic field generated by the permanent magnet in Example 2;

[0036] Figure 7 is a trend diagram of the linear motion of magnetic microbeads under the guidance of the permanent magnet in Example 2;

[0037] Figure 8 is a trend diagram of the magnetic microbeads passing through the cross section under the guidance of the permanent magnet in Example 2;

[0038] Fig. 9 This is a trend diagram of the linear motion of the magnetic microbeads when the position of the permanent magnet in Example 2 is fixed. DETAILED DESCRIPTION

[0039] For ease of understanding, the bionic vascular microchannel and its application for magnetic targeted delivery research are described below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0040] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation and positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0041] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0042] Example 1 Preparation of the bionic vascular microchannel for magnetic targeted delivery research

[0043] The bionic vascular microchannel used for magnetic targeted delivery research refers to the product obtained by replacing the chloroplasts and cell fluid in the plant leaves with deionized water.

[0044] The preparation method is as follows, which specifically comprises the following steps:

[0045] S11: In this embodiment, water spinach leaves are selected. Spinach leaves, Chinese cabbage leaves or sweet potato leaves with similar characteristics can also be selected. The samples with no damage on the leaf surface, complete appearance, high strength and an overall leaf length of 5-7 cm are selected. Figure 1 As shown in a, a mixed solution of exfoliating is prepared, specifically a mixed solution of 98% n-hexane aqueous solution and 1*PBS (phosphate buffered saline) in a volume ratio of 1:1, and the leaves are immersed in it to remove the leaf cuticle. After the treatment, the leaves are as follows Figure 1 As shown in b;

[0046] S12: The leaves treated with S11 were immersed in a 10g / 100ml SDS (sodium dodecyl sulfate) aqueous solution for about 120 hours, and then ultrasonically washed with a deionized water solution for 5 minutes. Figure 1 As shown in c;

[0047] S13: Soak the leaves treated in S12 in a prepared cell lysis mixed solution, specifically a deionized water solution of 0.1% Triton X-100 cell lysis solution and 10 wt% sodium chloride bleach mixed in a volume ratio of 1:1 for about 12 hours, and perform ultrasonic oscillation washing with the deionized water solution for 5 minutes. Figure 1 As shown in d;

[0048] S14: Use a 0.5 mm injection needle, the specific caliber selection can be adjusted according to the petiole size, and inject sterile deionized water from the petiole of the leaf. Use polyethylene raw tape or PTFE sealing water tape to seal the connection between the petiole and the needle. The injection pressure is controlled at about 1500 mmHg. Because there are many pore structures on the veins and leaves, the sterile deionized water is precipitated on the leaf surface under the injection pressure and gravity.

[0049] S15: The leaves treated with S14 were soaked in sterile deionized water at 4°C for two weeks. The experiment found that the transparency of the leaves after soaking was significantly improved compared with the leaves that were not soaked in low-temperature water. In order to facilitate the subsequent observation of the situation inside the leaf veins through a microscope, the leaves need to have sufficient transparency. Experimental verification shows that the soaking time must be at least 10 days. However, if the soaking time is too long, it must exceed 20 days. The leaf structure will become very soft and the strength will be greatly reduced, making it no longer suitable for subsequent operations.

[0050] The prepared leaves must be tested and screened before use. The specific testing method is as follows: Figure 2 As shown, the hanging drainage method is used until there is no water on the leaf surface; Figure 3 As shown in the figure, a syringe was used to inject the Ponceau aqueous solution from the petiole of the leaf to observe the integrity of the leaf and the patency of the veins. The morphology of the frozen section was observed under a microscope, as shown in the figure below. Figure 4 As shown, the microchannel structure formed inside can be seen.

[0051] Example 2 Magnetic field drives magnetic microspheres to move along a preset path in the bionic vascular microchannel used for magnetic targeted delivery research

[0052] S21: Use a microfluidic pump to infuse 10 ml of a magnetic microbead solution from the petiole of the leaf blade. In this embodiment, commercial agarose-coated ferroferric oxide nanoparticles are used. The specific product model is Affimagγ-Fe2O3, with a particle size of 3.0 μm-4.0 μm, and the surface group is a carboxylate ion. The concentration of the magnetic microbead solution is about 1% (g / 100 ml). It can also be micron-level immunomagnetic beads with a surface modified by hydroxyl or fluorescent light. Place the leaf with the magnetic microbeads on a transparent stage;

[0053] S22: Determine the movement trajectory of the magnetic beads, and obtain the relative position parameters of the magnetic beads and the guiding magnet when guiding the movement of the magnetic microbeads according to the movement formula of the magnetic beads when being pulled by the magnetic force in space, that is, the movement parameters of the guiding magnet. In this embodiment, a permanent magnet is used as the guiding magnet, and an electromagnetic coil or an electromagnetic coil group can also be used. In the process of controlling the movement of the magnetic microbeads, it is necessary to keep the leaf vein channel moist. The specific method can be as follows: Figure 5 As shown in a, place the sample in a wetted watch glass and rehydrate it regularly or Figure 5 As shown in b, covering the sample surface with a coverslip slows down the loss of water. During the driving process, an optical microscope is used to observe the movement of the magnetic microbeads for the study of magnetic targeted delivery.

[0054] The method for controlling the preset path of the magnetic microspheres in the above microchannel:

[0055] Assume that the permanent magnet is cylindrical, such as Figure 6 As shown, the vector position of the geometric center of the controlled magnetic microbead in the three-dimensional coordinate system is:

[0056]

[0057] The geometric center of a permanent magnet is the vector position in space. If it is an electromagnetic coil, it is the center position of the coil:

[0058]

[0059] The gradient magnetic field in the system is generated by the guide magnet, and its magnetization intensity is uniformly distributed along the z-axis. In the proposed system, the magnetic bar can be approximated as a dipole moment to simplify the calculation of the long-distance magnetic field intensity. Therefore, according to the first-order point dipole model, it can be obtained:

[0060]

[0061] Among them B G (P B ) is the point magnetic dipole P B The magnetic induction intensity at the point, μ0 refers to the spatial magnetic permeability, and the relative position between the magnetic microbead and the permanent magnet P relative =P B -P G , m G is the magnetic dipole moment of the permanent magnet, and T refers to the matrix torque.

[0062] In the process of the movement of the controlled magnetic microbeads guided by the permanent magnet, it can be assumed that the direction of the external magnetic field generated by the permanent magnet at the location of the magnetic microbead is the same as the magnetization direction of the magnetic microbead, that is, At this time, the magnetic force in the controlled magnetic microbead space is:

[0063]

[0064] Among them, M B is the magnetization intensity of the controlled magnetic microbeads, B G is the magnetic induction intensity of the permanent magnet.

[0065] Since the permanent magnet in this embodiment has cylindrical symmetry, magnetically driven motion control in the X and Y directions can be achieved in the same way. To simplify the analysis, this embodiment only analyzes the motion in the XZ plane, and the motion control implementation method in the Y direction refers to the X direction.

[0066] Only the displacement of the magnetic microbead and the permanent magnet in the same plane (xz plane) is considered, so the magnetic force F on the magnetic microbead M It can be expressed as:

[0067]

[0068] Among them, M Bx It refers to the magnetic moment component of the magnetic microbead in the X direction, M Bz It refers to the magnetic moment component of the magnetic microbead in the Z direction, B Gx It refers to the magnetic induction intensity component of the external magnetic field generated by the guiding magnet in the X direction, B Gz It refers to the magnetic induction intensity component of the external magnetic field generated by the guiding magnet in the Z direction.

[0069] Assume the relative position of magnetic beads and permanent magnet

[0070] Wherein w refers to the X component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead, and h refers to the Z component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead;

[0071] Then the kinematic equation of the magnetic microbeads when controlled by the permanent magnet is:

[0072]

[0073] Among them, F adh It refers to the viscous resistance of magnetic microbeads in a static liquid environment, which is obtained by the following equation:

[0074]

[0075] Where η refers to the viscosity of the fluid, r refers to the radius of the magnetic bead, It refers to the velocity component of the magnetic beads in the X direction, It refers to the velocity component of the magnetic microbead in the Z direction;

[0076] F bIt refers to the buoyancy of magnetic microbeads in a static liquid environment, which is obtained by the following equation:

[0077]

[0078] Where ρ is the liquid density, g is the acceleration constant, V B is the volume of the controlled magnetic microbeads;

[0079] F G Refers to the gravity exerted on the magnetic microbeads in a static liquid environment, which is obtained by the following equation:

[0080]

[0081] Where m B Refers to the mass of magnetic beads.

[0082] In summary, the motion state model of magnetic microbeads when pulled by magnetic force in space is:

[0083]

[0084] According to the above model, we can easily get that for any w, there is a corresponding where h balance It refers to the distance component in the Z direction between the magnetic beads and the guide magnet when the beads are in balance in the Z direction. It refers to the actual position height of the guiding magnet at this time, so that the magnetic beads maintain a force balance state in the Z direction. At this time, the magnetic beads receive the X-direction force of the permanent magnet and move at a variable speed on the X axis;

[0085] For any w, there exists a corresponding in It refers to the height position of the guide magnet, which is a function of w, causing the magnetic beads to move at a variable speed in the Z-axis direction.

[0086] Finally, by controlling the relative position parameters w and h of the magnetic microbeads-permanent magnets, the motion control of the magnetic microbeads in the XZ plane is achieved. Furthermore, PID and other control algorithms can be used to achieve more accurate control of the motion of the magnetic microbeads. The controlled magnetic microbeads are controlled by permanent magnets to achieve targeted delivery along the guided route in the leaf veins, that is, the bionic vascular microchannels.

[0087] like Figure 7 As shown, as the permanent magnet moves according to the motion parameters calculated by the motion formula, the magnetic microbeads are guided to perform linear motion in the bionic blood vessel microchannel structure, which confirms that the control method provided by the present invention can effectively control the linear motion of the magnetic microbeads, and the magnetic microbead group continues to move along the designed path.

[0088] like Figure 8 As shown, as the permanent magnet moves according to the motion parameters calculated by the motion formula, the magnetic microbeads are guided to move through the cross section in the bionic blood vessel microchannel structure, which confirms that the control method provided by the present invention can effectively control the magnetic microbeads to pass through the cross section, indicating that this motion model is still applicable when there is a cross section in the blade.

[0089] like Fig. 9 As shown, when the position of the permanent magnet is fixed, the magnetic microbeads are guided to move in the bionic vascular microchannel structure. It can be found that the magnetic microbeads will accumulate at the position of the permanent magnet, but cannot move along the designed movement path.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bionic vascular microchannel for magnetic targeted delivery research, characterized in that: It refers to the product obtained by replacing the chloroplasts and cell fluid in the leaves of the plant with deionized water.

2. The bionic vascular microchannel for magnetic targeted delivery research according to claim 1, characterized in that: The plant leaves are selected from water spinach leaves, spinach leaves, Chinese cabbage leaves or sweet potato leaves.

3. The bionic vascular microchannel for magnetic targeted delivery research according to claim 1, characterized in that: The bionic vascular microchannel used for magnetic targeted delivery research is prepared by the following method, which specifically includes the following steps: S11: Select a suitable leaf and remove the cuticle of the leaf using an exfoliating mixed solution; S12: Soak the leaves treated by S11 in a deproteinization solution for 100-140 hours, then wash and proceed to the next step; S13: Soak the leaves treated with S12 in the prepared cell lysis mixed solution for 10-14 hours, then wash and proceed to the next step; S14: Infuse sterile deionized water from the petiole of the leaf, and control the infusion pressure at 120- 180mmHg, until water precipitates from the leaf surface; S15: Soak the leaves treated with S14 in sterile deionized water at 3-5°C for 10-20 days and then take them out for testing.

4. The bionic vascular microchannel for magnetic targeted delivery research according to claim 3, characterized in that: In step S15, the temperature of the sterile deionized water is 4° C., and the duration is preferably 13-15 days.

5. The bionic vascular microchannel for magnetic targeted delivery research according to claim 3, characterized in that: The detection method in step S15 is: firstly, the leaves are hung and drained until there is no water on the leaf surface, and then a Ponceau aqueous solution is used to perfuse from the petiole to observe the integrity of the leaves and the patency of the veins.

6. A control method for driving magnetic microspheres to move along a preset path in a bionic vascular microchannel for magnetic targeted delivery research as described in any one of claims 1 to 5 using a magnetic field, specifically comprising the following steps: S21: Use a microfluidic pump to infuse 8-12 ml of a 0.1-1% (g / 100 ml) magnetic microbead solution from the petiole of the leaf blade, and place the sample into which the magnetic microbeads have been introduced on a transparent stage; S22: According to the motion state model of the magnetic microbeads when being pulled by magnetic force in space, the adjustment data of the center position of the guiding magnet is obtained, thereby realizing the directional motion of the magnetic microbeads and observing the motion process of the magnetic microbeads using an optical microscope.

7. The control method according to claim 6, characterized in that: The magnetic microbeads are micron-sized microbeads with ferroferric oxide nanoparticles wrapped in agarose or micron-sized immunomagnetic beads with surfaces modified by hydroxyl groups or fluorescent lamps.

8. The control method according to claim 6, characterized in that: When the magnetic microbeads are pulled by magnetic force in space, the motion state model in the XZ plane is as follows: in, It refers to the magnetic field force exerted on the magnetic beads in the X direction. It refers to the magnetic field force on the magnetic beads in the Z direction, m B refers to the mass of the magnetic microbeads, 6πηr refers to the fluid resistance of the magnetic microbeads, It refers to the speed of the magnetic beads in the X direction. refers to the speed of the magnetic microbeads in the Z direction, w refers to the X component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead, and h refers to the Z component of the distance between the end center point of the guiding magnet and the mass center of the controlled magnetic microbead; For any w, there exists a corresponding where h balance It refers to the distance component in the Z direction between the magnetic beads and the guide magnet when the beads are in balance in the Z direction. It refers to the actual position height of the guiding magnet at this time, so that the magnetic beads maintain a force balance state in the Z direction. At this time, the magnetic beads receive the X-direction force of the permanent magnet and move at a variable speed on the X axis; For any w, there exists a corresponding in It refers to the height position of the guiding magnet, which is a function of w, causing the magnetic beads to move at a variable speed in the Z-axis direction.

9. The control method according to claim 8, characterized in that: The guiding magnet is formed by a permanent magnet, an electromagnetic coil or an electromagnetic coil group. When the guiding magnet is a permanent magnet, its center position is the geometric center; when the guiding magnet is an electromagnetic coil or an electromagnetic coil group, its center position is the center of the coil or the coil group.

10. The control method according to claim 8, characterized in that: When performing step S22, the leaf vein channel needs to be kept moist, the sample is placed in a moistened surface dish and water is added regularly or a cover glass is covered on the surface of the sample.

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