KR101765070B1 - Method and device for differentiation of cell using analyzing cell's movements - Google Patents

Abstract

The present invention relates to a new method and apparatus for mesoscopic biological particle discrimination that analyze the statistical movement of particles associated with structural characteristics and physical interactions of mesoscopic biological particles, The present invention relates to a cell differentiation method comprising observing and analyzing random movement of cells and a glassy substrate for positioning the cells.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for distinguishing a cell from a cell,

The present invention includes the steps of placing the stereoscopic meso biological particles (such as cells) on a glass substrate (for example, SiO _2.); And a, meso stereoscopic biological particles containing the (SiO ₂ for example.), Meso stereoscopic biological particle distinction method and meso stereoscopic biological particles are glass substrate for position, comprising the step of observing and analyzing the random movement of the meso stereoscopic biological particle And a mechanism for random motion observation.

There is a cell-observing device for optically observing cells (Kanegasaki S. et al.). This mechanism is constituted of, for example, a glass plate and a silicon chip having an engraved surface formed by etching so as to constitute a cell receiving area between the opposed surfaces of the opposed surfaces. The cells are dispersed in a cell accommodating space in a planar manner and can be observed through an optical observation apparatus such as a microscope through a glass plate. By using this cell observation device, for example, it is possible to observe the exercise capacity of the cell, the response to the stimulating substance, and the influence of the concentration gradient of the stimulating substance.

Japanese Patent Publication No. 2747304 discloses a technique for observing cells when the cells are dispersed in the observation field of view and placed in a fixed state at each position for observation. This technique is to arrange and fix the cells on the observation plane by providing the through holes for aspiration on one side of the face member constituting the cell accommodation space and connecting the through holes to the suction device and sucking the cells.

On the other hand, a technique for observing a cell for observing the position without changing the position without damaging the cell is disclosed in Korean Patent Laid-Open Publication No. 2007-0116112, which affects the flow of the liquid filling the space for accommodating cells and diffusion of the substance And it is possible to observe the cells in a state in which they are arranged in a reproducible manner at a desired position without imposing a large load on the cells.

Japanese Patent Publication No. 2747304 Korea Patent Publication No. 2007-0116112

Kanegasaki S et al., (2003) J. Immunol. Methods 282, 1-11

The present invention seeks to provide a new method for analyzing the statistical movement of particles (e.g., cells) associated with structural characteristics and physical interactions of mesoscopic biological particles, and a mechanism for mesoscopic biological particle discrimination.

The present invention includes the steps of placing the cell on a glass substrate (for example, SiO _2.); And observing and analyzing the random movement of the cells, comprising a cell differentiation method and a glassy substrate (e.g., SiO ₂ ) for the cell to locate, .

In the present invention, it was found that cPSMS motion is normal diffusion on a substrate having a negative charge. This is due to the electrostatic repulsion between the charged cPSMS and the substrate . In addition, spherical living cells were observed on a substrate with negative charge, and observed to move like cPSMS. However, MSD measurements showed that spherical living cells exhibited super-diffusive behavior. The anomalous movement of these cells seems to be due to the rocking motion of cells in random walk, which is due to the mechanical properties (ie resilience, resilience) of the cells as well as the surface heterogeneity of the cells It seems to be due to physical interaction. Experiments have shown that the random movement of cells (ie, resilient and resilient spheres) is affected by non-specific binding to locally and temporarily formed substrates induced by heterogeneity of cell surface charge, The present invention can provide a method of observing the behavior of a cell in the absence of an external force, for example, a chemical concentration gradient, pressure, or electrical stimulation, There is a technical significance in that the cells can be distinguished from dead cells.

Figure 1 is a graphical representation of the process of statistical movement of particles due to electrostatic interactions between spherical mesoscopic particles and a glassy substrate (SiO ₂ ), wherein (b) and (c) Typical steps are shown.
FIG. 2A shows the measured mean moving speed, FIG. 2B shows the MSD of living cells and cPSMS, FIG. 2C shows the result of locus analysis obtained from 100 particles, and the displacement of living B16F10 cells (green) and cPSMS The PDF is represented by an optimal-fit curve by a Gaussian pit (dashed line).
3A shows a method of extracting an angle value q from a random motion locus of a particle according to a time flow t and FIG. 3B shows a method of extracting an angular distribution of a random work locus of living B16F10 cells (upper) and cPSMS (lower) Fig.
(C) - (f) shows the random motion locus (c) of the dead cells. FIG. 4A is an optical image of a cell showing dead cells stained with trypan blue, , Average moving speed (d), MSD (e) and angular distribution (f).
FIG. 5 is a diagram showing how a cell vibrates due to electrostatic interaction between a cell and a glassy substrate.

The present invention relates to a method for the preparation of mesoscopic biological particles, comprising the steps of: placing mesoscopic biological particles on a glassy substrate; And analyzing the statistical movement of the particles. ≪ RTI ID = 0.0 > [0002] < / RTI >

The present invention provides a method for preparing a cell, comprising: positioning a cell on a glassy substrate; And observing a random walk of the cells.

In one aspect of the present invention, the glass substrate, for example, SiO _2, Si, Si ₃ N _4, mica not necessarily be (mica) can but be limited to, silicates, rod-silicate, phosphate, zinc-rod silicate, Soda-lime-silica, lead-silicate and lead-zinc-borate glassy substrates, and more specifically SiO ₂ (glassy silicon dioxide) may be used.

In one aspect of the invention, the mesoscopic biological particle can be a cell.

In one aspect of the invention, a glassy substrate is formed on a silicon wafer.

In one aspect of the invention, a silicon dioxide glassy layer is formed on a silicon wafer.

In one embodiment of the invention, the glassy substrate is 0.1 to 20 kA, specifically 1 to 10 kA, more specifically 5 kA.

In one aspect of the invention, the vitreous substrate is a silicon dioxide glassy layer.

In one aspect of the invention, polydimethylsiloxane as a reservoir may be additionally located on the glassy substrate, but not limited thereto, since the movement of the microspheres must be observed on a glassy substrate in the presence of a solution, Other mold types that can be contained, for example, plastic resin, metal, PDMS other than silicone, paraffin, etc. may also be used.

In one embodiment of the present invention, the thickness of the polydimethylsiloxane reservoir may be 0.1 to 20 mm.

In one aspect of the invention, the observation and analysis of the mesoscopic biological particles (e. G., Cells) can be used to distinguish whether the mesoscopic biological particles are alive or dead.

In one aspect of the invention, when mesoscopic biological particles (e. G., Cells) are alive, Brownian motion can be observed due to electrostatic repulsion between the mesoscopic biological particles and the substrate.

In one aspect of the invention, mesoscopic biological particles (e.g., cells) can exhibit super-diffusivity as a result of MSD measurements when live.

The present invention also relates to a mechanism for random motion observation of mesoscopic biological particles, wherein the mesoscopic biological particles consist of a vitreous substrate for positioning.

In one aspect of the invention, it relates to a mechanism for random motion observation of mesoscopic biological particles comprising a vitreous substrate (e.g., SiO ₂ ) for cell placement.

In one aspect of the present invention, the glass substrate, for example, SiO _2, Si, Si ₃ N _4, mica not necessarily be (mica) can but be limited to, silicates, rod-silicate, phosphate, zinc-rod silicate, Soda-lime-silica, lead-silicate and lead-zinc-borate glassy substrates, and more specifically SiO ₂ (glassy silicon dioxide) may be used.

In one aspect of the invention, the mesoscopic biological particle can be a cell.

In one aspect of the invention, a glassy substrate is formed on a silicon wafer.

In one aspect of the invention, a silicon dioxide glassy layer is formed on a silicon wafer.

In one embodiment of the invention, the glassy substrate is 0.1 to 20 kA, specifically 1 to 10 kA, more specifically 5 kA.

In one aspect of the invention, the vitreous substrate is a silicon dioxide glassy layer.

In one aspect of the present invention, polydimethylsiloxane as a reservoir is additionally located on the glassy substrate.

In one embodiment of the present invention, the thickness of the polydimethylsiloxane reservoir may be 0.1 to 20 mm.

<Abbreviation Definition>

MSD: mean square displacements

cPSMS: carboxylated polystyrene microspheres

< Experimental Example  >

1. Preparation of cells and negatively charged artificial particles

B16F10 cells (ATCC, USA) for use in random work observation were treated with trypsin EDTA (20 w / w%) for 3 minutes to separate and obtain a spherical shape (diameter ~ 16 μm) in the suspension. Residual trypsin EDTA was immediately removed by rinsing with buffer (8.6 w / w% sucrose, 0.3 w / w% glucose and 1.0 mg / mL bovine serum albumin, pH 5.2). Cells were resuspended in buffer for the next experiment. To demonstrate the stochastic process of cells, dead cells were used and cells stained with trypan blue were identified by hemocytometer. To avoid the effects of staining molecules in stochastic processes, cells were not stained in random work tests. The cPSMS (15 [mu] M, Kisker-Biotech, Germany) used for random work experiments was diluted in the same buffer (~ 250 [mu] g / mL).

2. Experimental process

To fabricate a microfluidic device with a negatively charged substrate, a silicon dioxide glassy layer (~ 8 kA) was grown on silicon wafer 100 by plasma chemical vapor deposition. The substrate was washed with piranha solution and solvent cleaning. A reservoir (~ 200 μm thick) made of polydimethylsiloxane was placed on the glassy substrate. A solution containing carboxylated polystyrene microspheres (cPSMS) or live cells (B16F10) was introduced into the reservoir and the reservoir top was covered with a glass slide to prevent the solution from evaporating. The motion of the two particles (B16F10 cell, cPSMS) was then recorded at 30 frames / s using a charge coupled device (CCD) camera. 100 trajectory abnormalities of B16F10 cells and cPSMS were collected and statistically reliable data were obtained. Particle tracking was performed with image analysis software (ImageJ 1.47v, NIH, USA) and sub-micron particle tracking was performed. For the MSD analysis, the χ-displacement of the trajectory was used and the calculation based on the y-displacement showed the same result.

<Experimental Results>

1. Charged Microsphere Tracking

Two-dimensional random motion of charged microspheres (B16F10 cells and cPSMS) was observed with a microfluidic device (see FIG. 1A). The cells were found to have a diameter of 16.2 ± 1.7 μm (see FIG. 1b). The behavior of both microspheres appeared similar to a conventional random walk and moved several tens of micrometers within a few square micrometers (~ 3 × 3 μm ² ) until observation time ~ 33 seconds (see FIGS. 1B and 1C). The biggest difference between cells and cPSMS is the overlap of random motion patterns, and living cells tend to return to the same place a few times. The weak force that affects the random walk was the electrostatic repulsion between the negatively charged microspheres (including cells) and the vitreous surface.

Conversely, the effect of electrostatic attraction between the microsphere and the vitreous surface was evaluated, and the PSMS functionalized with an amino group (positively charged in a given solution) exhibited a very strong electrostatic charge between the positively charged amine-PSMS and the negatively charged glassy substrate It was confirmed that the workforce did not perform any movement on the substrate.

Spherical B16F10 cells were observed for a prolonged period of time for 90 minutes to see if the random movement during the given time (~ 33 seconds) was due to roughness fluctuations due to the glassy substrate. Most cells exhibited large scale displacements (~ 35 μm) at any given time, and the movement of cells by thermal agitation diffused rather than only on the spot, and the normal time scale ~ 33 seconds) was enough to observe and analyze the movement of these cells.

2. Random Walk  Statistical analysis

) 의 변위로부터 계산되었다.To understand the baseline information on the trajectory, the average track velocity (v) was calculated from the locus of live B16F10 cells and cPSMS (see Figure 2a). In particular, ν is the number of steps (ie,

). &Lt; / RTI &gt;

Experimental results show that ν is dependent on τ (here, 33 ms) and the displacement (r) at a given time interval (τ) is due to the fact that it is not very large. Interestingly, living cells were about three times faster than sPSMS (ie, ν _B16F10 / ν _cPSMS ~ 3). In addition, the histogram of ν _B16F10 was wider than that of cPSMS. The faster motion and the larger standard deviation of v _B16F10 mean that there is more complex interaction between the cell and the substrate than cPSMS. To explore another characteristic of the stochastic process, cPSMS and one-dimensional MSD of living cells were calculated based on measurements (see FIG. 2b).

0.024 μm²·s^-1이었다. 이러한 결과는 스톡스-아인슈타인 관계식, D=k_BT/6 πμr, 여기에서 k_B는 볼츠만 상수이고, T는 온도이며, μ은 점성계수(물에 대한), r은 마이크로스피어의 직경이고, 상기 식으로부터 도출된 D 값 (

0.028 μm²·s^-1)과 일치한다. 살아있는 세포의 경우 그러나 MSD는 <△x²(t)> ~ t^γ로 표시되는 레비 워크 모델에 부합하였고, 여기에서 γ는 랜덤 워크의 특징을 결정짓는 파라미터이다. B16F10 살아있는 세포의 MSD는 t^γ에 비례했고, γ

1.5 (즉, 수퍼-확산성)으로 나타났다. The calculated MSD of cPSMS appeared to follow the normal diffusion model, whereas random motion of living cells was dominated by super-diffusion behavior instead. In particular, the cPSMS MSD is well beaten in Einstein's equation ^{<△ x 2 (t)>} = 2Dt, and herein D indicates a diffusion coefficient, D in cPSMS

0.024 μm ² · s ^-1 . This result is the Stokes-Einstein equation, and D = k _B T / 6 πμr, where k _B is Boltzmann's constant, and T is temperature, μ is viscosity, and (in water), r is the diameter of the microsphere, the D value derived from the equation (

0.028 μm ² · s ^-1 ). In the case of living cells, however, the MSD matched the Leviwalk model, denoted by △ x ² (t)> ~ t ^γ , where γ is a parameter that determines the characteristics of the random walk. The MSD of B16F10 live cells was proportional to t [ ^gamma] , and [gamma]

1.5 (i.e., super-diffusive).

We also obtained the probability density function (PDF) of cPSMS and χ-displacement of living cells (see Figure 2c). PDF of both microspheres (ie, B16F10 and cPSMS), although the PDF of living cells has a heavey tail compared to cPSMS, the microscopic displacement f (x, t) ~ exp (-x ² / 4Dt). &Lt; / RTI &gt; This difference means that the random walk of B16F10 live cells is also a super-diffusion process, consistent with the MSD tendency (see FIG. 2b).

Therefore, according to the MSD and PDF analysis, it can be seen that the random walk of cPSMS follows the normal spread, whereas the B16F10 live cells follow the super-diffusion process. However, no long step corresponding to Levy walk was observed in the random walk trace of the cells (see FIG. 1 b). These conflicting results have shown that conventional methods (ie, MSD or PDF) are insufficient to fully understand the anomalous movement of living cells.

In order to observe more closely the characteristics of the random walk of B16F10, the distribution of relative angles ( ? ) Of motion between consecutive time intervals of the random work locus was analyzed (see FIG. These parameters have recently been used to understand the anomalous random walk of proteins or biological fibrils. The direction of both microspheres moving on the substrate can be changed from -π to pi. As expected, the distribution of θ derived from the trajectory of cPSMS was uniform from -π to π, while U-shaped patterns were shown for living cells (see FIG. 3b). Of course, there was no chemical concentration gradient because no chemicals (eg, cytokines) were used in this experiment. The anisotropy of the angular distribution is seen as a result of the presence of the physical field (i.e., weak binding force or rheological properties of the cell). The symmetric regime is a statistical trend associated with the trajectory of hundreds of cells. In particular, the trajectory of a single cell was not symmetric but showed anisotropy.

1.5 × 10^-6) 표시되는 다항 피팅 그래프로 이차 다항 상수(quadratic polynomial coefficient) (α)값을 도출하였다. 이러한 경향은 cPSMS과는 달리 대부분의 살아있는 세포에서 관찰한 시간 간격에서 그 경로가 급격히 변화하는 양상을 보였다. 특히, 불균형한 상황에서 균형을 맞추려고 할 때, (예. 진동 모션(rocking motion)) 롤리-폴리 차임 볼(roly-poly chime ball)과 같은 지그재그 패턴으로 움직일 수 있다. 이러한 모션은 상기 기재된 B16F10 살아있는 세포의 변칙적 운동과 밀접하게 관련되어 있다고 생각된다. 만약 세포들이 열 교반(thermal agitation)에 대한 반응으로 랜덤하게 움직인다면, 진동 모션을 나타낼 것이고 그 운동의 총 변위는 증가할 것이며, 상이한 확산 형태(diffusion regime)를 나타낼 것이다. 상기 진동 모션은 세포들과 cPSMS 사이의 구조적 차이에 기인할 수 있다. cPSMS는 균질한 고체 구형인 반면 세포는 몇 kPa의 소프트하고, 탄력성이 있는 구조적 특징을 갖는 세포골격을 갖는다. B16F10 세포가 기판으로부터 분리될 때 구형으로 보인다 하더라도, 그들은 외부 및/또는 내부 에너지가 세포의 항상성을 유지하는 데 필요하기 때문에 물-젤리와 같이 미세하게 변경될 수 있다. 또한 막 단백질과 같은 막 구성성분을 포함하는 복잡한 이온 교환이 살아있는 세포의 표면 전하의 불균질성을 야기할 수 있다. 이러한 불균질성은 세포 막과 유리 기판 사이에 비특이적인 약한 결합력(예를 들어 반 데르 발스 힘)을 유도할 수 있고, 상이한 운동 특징을 나타내게 만든다.
To measure the degree of U-shape distribution, P ( θ ) ~ α θ ² (? _B16F10

1.5 × 10 ^-6 ) quadratic polynomial coefficient (α) was derived from the displayed polynomial fitting graph. Unlike cPSMS, this tendency shows that the pathway changes rapidly at the time interval observed in most living cells. In particular, when trying to balance in an unbalanced situation, it can be moved in a zigzag pattern, such as a roly-poly chime ball (eg rocking motion). These motions are thought to be closely related to the anomalous movement of the B16F10 live cells described above. If cells move randomly in response to thermal agitation, they will exhibit vibrational motion and the total displacement of the motion will increase and will exhibit a different diffusion regime. The vibrational motion may be due to structural differences between the cells and the cPSMS. cPSMS is a homogeneous solid sphere, while cells have a cytoskeleton with soft, elastic structural characteristics of a few kPa. Although B16F10 cells appear spherical when they are detached from the substrate, they can be minutely altered as water-jelly because external and / or internal energy is required to maintain cell homeostasis. Complex ion exchange, including membrane constituents such as membrane proteins, can also cause heterogeneity in the surface charge of living cells. This heterogeneity can lead to nonspecific weak binding forces (e. G., Van der Waals forces) between the cell membrane and the glass substrate, resulting in different kinetic characteristics.

3. Observation and analysis of dead cell movement

1.95 ± 0.13 μm·s^-1이고, γ_{d e ad cell}

1.3)(도 4c-4e 참조). 일반적으로, 죽은 세포들은 적은 질량을 가졌고 따라서 모멘텀 보존의 법칙에 따라 더 빠른 속도를 갖는다. 그러나 죽은 세포의 평균 속도는 살아있는 세포의 54% 미만이고, 이는 입자의 질량이 적어도 죽은 세포의 랜덤 워크의 스토캐스틱 파라미터를 감소시키는 중요한 인자는 아니라는 점을 시사한다. 도한 세포의 감소된 질량은 아마도 그들의 기계적 물성을 경직시키는 것과 연관이 있을 것이다. 낮은 물 함량 때문에 죽은 세포는 물리학적으로 회복력이 떨어질 것으로 예상되고, cPSMS 고체형과 같이 더 행동할 것으로 예상되며, 이는 세포의 유리질-유사 운동역학(예를 들어 세포골격의 유리 전이) 또는 세포성 텐세그리티(tensegrity) 모델과 관련이 있을 것으로 보인다. 또한 죽은 세포에서는 이온 교환이 일어나지 않으며 따라서 표면 전하 불균질성은 살아있는 세포에 비해 낮을 수 밖에 없다. 이러한 차이점으로 인하여 죽은 세포 및 기판 사이의 상호작용이 더 적을 것으로 예상된다. To further investigate why cells exhibit a different pattern from cPSMS, we made dead cells through starvation (see FIGS. 4a and 4b) and observed the movement pattern. Most dead cells made by starvation seem to retain their shape (eg, spherical) (Fig. 4A). The parameter values that characterize the random walk of dead cells were changed from live cells to cPSMS. (v _{dead cell}

1.95 ± 0.13 μm · s ^-1 , γ _{d e ad cell}

1.3) (see Figures 4c-4e). In general, dead cells have a low mass and therefore have a faster rate according to the law of conservation of momentum. However, the mean rate of dead cells is less than 54% of living cells, suggesting that the mass of the particles is not an important factor to at least reduce the stochastic parameters of random walk of dead cells. Reduced mass of cells will probably be associated with stiffening their mechanical properties. Due to the low water content, dead cells are expected to be physically less resilient and are expected to behave more like cPSMS solid forms, which may be due to the glassy-like kinetic dynamics of the cell (eg, glass transition of the cytoskeleton) It seems to be related to the tensegrity model. Also, ion exchange does not occur in dead cells, and surface charge heterogeneity is inferior to living cells. This difference is expected to result in less interaction between the dead cells and the substrate.

7.6 × 10^-7). 이러한 실험 결과를 통해, 세포성 진동 모션(cellular rocking motion)이 상대적으로 적다는 점이 죽은 세포의 궤적에도 또한 보여진다는 점을 알 수 있다(도 4c). 이러한 각도 분포의 변화는 진동 모션을 포함하는 세포의 변칙적 운동이 세포의 특성(회복력, resilience)뿐만 아니라 표면 전하 불균질성에 기인하는 것이라는 가설을 뒷받침한다. 이러한 현상을 도 5에 도식화하였다. 표면 전하의 불균질성은 예를 들어 단백질과 같은 막 구성성분(즉 전하를 띈 분자)뿐만 아니라 당단백으로부터 유래하며, 세포의 비정상적인 운동을 유도하는 국소적인, 일시적인 제한에 있어서 중요한 인자가 될 수 있다. 이러한 불균질성은 살아있는 세포와 기판 사이의 비특이적 결합에 영향을 미치며, 변칙적 랜덤 워크를 야기한다.
Although there are subtle differences in angular distribution between living and dead cells, angular distribution of dead cells indicates a U-shaped pattern. The positive peaks near -π and -π in the U-shaped pattern were smaller in dead cells but did not disappear (α _{dead cell}

7.6 × 10 ^-7 ). From these experimental results, it can be seen that the relatively low cellular rocking motion is also seen in the trajectory of dead cells (FIG. 4c). This change in angular distribution supports the hypothesis that the anomalous movement of cells, including vibrational motion, is due to the surface charge inhomogeneity as well as the cell properties (resilience, resilience). This phenomenon is illustrated in FIG. The heterogeneity of surface charge can be an important factor in the local, transient limitation leading to abnormal cell movement, for example, from membrane components such as proteins (ie charged molecules) as well as glycoproteins. This heterogeneity affects nonspecific binding between living cells and the substrate, resulting in an irregular random walk.

<Conclusion>

Observation of the random walk of spherical living cells shows that cPSMS is normal diffusion although moving on a substrate (ie moving in a two-dimensional region, not in a three-dimensional solution space) This seems to be due to the Brownian motion when the charged microspheres produce an electrostatic repulsion between them and the substrate.

In addition, MSD measurement results showed that spherical living cells exhibited super-diffusive behavior. The anomalous movement of these cells appears to be due to the rocking motion of the cells in random walk, which is not only due to the mechanical properties of the cell (ie, resilience, resilience), but also the physical interaction by cell surface charge heterogeneity . &Lt; / RTI &gt; This theory has been demonstrated by random walk analysis of dead cells. In the case of cells through experiments (ie, resilient and flexible spheres), random motion is not only due to transient weak confinement of the cell membrane, but also due to non-specific binding between cells and substrates induced by cell surface charge heterogeneity And can be affected by subtle deformation.

Thus, the present invention can provide a way to look at the behavior of cells in the absence of external forces, such as chemical concentration gradients, pressure or electrical stimulation, and to distinguish between living and dead cells without a process such as cell staining It is significant in that it can be.

Claims (21)

Placing mesoscopic biological particles on a glassy substrate formed on a silicon wafer; And
And analyzing the statistical movement of the particle,
Here, the mesoscopic biological particle is a cell,
Polydimethylsiloxane as a reservoir is additionally located on the glassy substrate,
In the case of living cells, Brownian motion is caused by the electrostatic repulsion between the cells and the substrate. As a result of MSD (mean square displacements) measurement,
Through analysis, it is possible to distinguish whether mesoscopic biological particles are alive or dead,
A method for distinguishing mesoscopic biological particles.
delete The method according to claim 1,
The vitreous substrate can be formed of a group consisting of SiO ₂ , Si, Si ₃ N ₄ , mica, silicates, bimosilicates, phosphates, zinc-bosilicates, soda-lime-silica, lead-silicates and lead-zinc- &Lt; / RTI &gt; wherein the mesoporous material is selected from the group consisting &lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;
The method according to claim 1,
Characterized in that the glassy substrate is a SiO ₂ (silicon dioxide) substrate.
delete The method according to claim 1,
Wherein the glassy substrate is between 0.1 and 20 &lt; RTI ID = 0.0 &gt; kA. &Lt; / RTI &gt;
The method according to claim 1,
Wherein the glassy substrate is between 1 and 10 &lt; RTI ID = 0.0 &gt; kA. &Lt; / RTI &gt;
delete The method according to claim 1,
Characterized in that the thickness of the polydimethylsiloxane reservoir is 0.1 to 20 mm.
delete delete delete A glassy substrate for positioning the mesoscopic biological particles,
The glassy substrate is formed on a silicon wafer,
Wherein the mesoscopic biological particle is a cell,
Polydimethylsiloxane as a reservoir is additionally located on the glassy substrate,
In the case of living cells, Brownian motion is caused by the electrostatic repulsion between the cells and the substrate. As a result of MSD (mean square displacements) measurement,
An instrument for random motion observation of mesoscopic biological particles capable of distinguishing whether the mesoscopic biological particles are alive or dead through analysis.
14. The method of claim 13,
Characterized in that the mesoscopic biological particle is a cell.
14. The method of claim 13,
The vitreous substrate can be formed of a group consisting of SiO ₂ , Si, Si ₃ N ₄ , mica, silicates, bimosilicates, phosphates, zinc-bosilicates, soda-lime-silica, lead-silicates and lead-zinc- &Lt; / RTI &gt; The apparatus of claim 1, wherein the mesoscopic biological particles are selected from the group consisting of:
14. The method of claim 13,
Characterized in that the vitreous substrate is a SiO ₂ (silicon dioxide) substrate.
delete 14. The method of claim 13,
Characterized in that the glassy substrate is between 0.1 and 20 &lt; RTI ID = 0.0 &gt; kA. &Lt; / RTI &gt;
14. The method of claim 13,
Characterized in that the glassy substrate is between 1 and 10 &lt; RTI ID = 0.0 &gt; kA. &Lt; / RTI &gt;
delete 14. The method of claim 13,
Characterized in that the polydimethylsiloxane reservoir has a thickness of 0.1 to 20 mm.

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