A method for simultaneous measurement of water and dry mass of living cells

Abstract

The present application belongs to the technical field of quality measurement, and particularly relates to a method for synchronously measuring water content quality and dry quality of living cells. The method comprises the following steps: capturing a cell to be measured by an optical tweezer; illuminating the cell to be measured based on a probe light source, so that the probe light is divided into backscattered light and forward scattered light; measuring a motion trajectory of the cell to be measured by using the backscattered light, and processing the motion trajectory into an instantaneous velocity power spectral density; generating a spatial distribution of phase delay of cell contents to the probe light by using the forward scattered light, and processing the spatial distribution into a phase shift distribution of the probe light after penetrating through the cell; fitting the instantaneous velocity power spectral density by using a limited Brownian motion theory of particles with fluid memory effect, and calculating the water content quality of the cell; and obtaining the dry quality of the cell by two-dimensional integration within a cell profile based on a linear relationship between a non-water content density and a refractive index increment. The present application realizes non-invasive, non-contact and synchronous measurement of the water content quality and the dry quality of living cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of quality measurement, and particularly relates to a method for synchronously measuring water content quality and dry quality of living cells. BACKGROUND

[0002] The physical properties of single cells, such as mass, volume and density, are important indicators of cell metabolic characteristics and homeostasis. Studies have shown that the physical properties of single cells, such as mass, volume and density, can not only characterize the different stages of the cycle, but also to a large extent reflect the basic life characteristics of cells, such as metabolic rate, reproduction, cell growth or proliferation, apoptosis and necrosis, mitosis and drug response. Similarly, these physical properties also help people better understand many diseases and accelerate the development of new drugs that affect the metabolism and development of the entire animal. Some chronic diseases, such as cancer and tumors, affect the physiological properties in cells, resulting in changes in cell mass and density. Therefore, the physical properties of single cells, such as mass and volume, provide key indicators and new ways for the diagnosis and treatment of diseases. However, due to the small volume of single cells, accurate measurement of their mass has always been a challenge.

[0003] Currently, the typical methods for detecting the mass of single cells are as follows:

[0004] Single-cell mass spectrometry, in which mass spectrometry is used to quantitatively detect various metal markers in each cell and understand the content of each target protein in each cell. It has the ability of high-speed analysis and high-resolution mass spectrometry. Single-cell mass spectrometry flow cytometry can simultaneously analyze more than 40 cell parameters at the single-cell level, greatly improving its ability to evaluate complex cell systems and processes. However, this method is difficult to use for measuring the overall mass of single cells.

[0005] Micro-mechanical method, in which the resonance frequency shift of microstructures is converted into the buoyant mass of cells. Suspended microchannel resonator (SMR) is a resonator device with microfluidic channels in the micro-mechanical method. Suspended microchannel resonator is beneficial to high-precision measurement of single-cell mass due to its small volume, low cost and high sensitivity, and has been widely studied and applied in the field of biochemistry. However, this method is difficult to integrate with optical structures such as imaging microscopes, making it difficult to achieve long-term observation of cell growth, and cannot be used to measure the dry mass of cells.

[0006] Magnetic levitation image cytometry, in which magnetic levitation technology is used to levitate cells, and single-cell parameters can be calculated through microfluidic and image processing techniques. This device is fully compatible with upright or inverted fluorescence microscopes, and can achieve real-time, label-free separation and high-resolution monitoring of cells. Similarly, this technology is difficult to measure the dry mass of cells.

[0007] Quantitative phase imaging (QPI) technology, which realizes non-contact and non-invasive single-cell dry mass measurement through quantitative phase reconstruction. This method is not limited by cell type and can be used in combination with other imaging techniques. However, this method cannot obtain the water content of the cell.

[0008] Therefore, the above methods are difficult to meet the requirements of not limiting the environment of the cell and simultaneously obtaining the water content and other content information of the cell. Therefore, there is an urgent need for a method that does not change the environment of the cell, can simultaneously measure the dry mass and water content of the cell, and is compatible with optical microscopes and other devices. SUMMARY

[0009] To solve the technical problems existing in the prior art, the present application provides a method for simultaneously measuring the water content and dry mass of living cells. After imaging the cells, the cells are captured by an optical tweezer to make restricted Brownian motion in the center of the optical trap. The motion trajectory of the cells and the spatial distribution of the phase delay of the probe light by the cell contents are measured at high speed. The data is processed in real time to obtain the real-time water content and dry mass of the cells. This method does not change the environment of the object and is non-contact to the cell.

[0010] To achieve the above object, the present application is realized by the following technical scheme:

[0011] A method for simultaneously measuring the water content and dry mass of living cells, which uses an optical tweezer capture device and a multi-parameter measurement device; the method comprises the following steps:

[0012] (1) capturing the cell to be measured by the optical tweezer capture device, so that it is bound in the optical trap to make restricted Brownian motion;

[0013] (2) illuminating the cell to be measured by a probe light source based on a multi-parameter measurement device, so that the probe light is divided into backscattered light and forward scattered light by the cell to be measured;

[0014] (3) measuring the motion trajectory of the cell to be measured making restricted Brownian motion at a preset measurement time resolution using backscattered light; generating the spatial distribution of the phase delay of the probe light by the cell contents using forward scattered light;

[0015] (4) performing fast Fourier transform on the trajectory of the cell to be measured to obtain the instantaneous velocity power spectral density; performing real-time processing on the spatial distribution of the phase delay of the probe light to extract the phase shift change data of each point to obtain the phase shift distribution of the probe light after passing through the cell;

[0016] (5) fitting the instantaneous velocity power spectral density by the theory of restricted Brownian motion of microparticles through the fluid memory effect, calculating the water content of the cell; based on the linear relationship between the non-water content density and the refractive index increment, obtaining the dry mass of the cell through two-dimensional integration within the cell profile.

[0017] Further, in the optical tweezer capturing device, the first light beam emitting device is a continuous wave single frequency laser with a wavelength in the blue-green spectrum range of 400-600 nm.

[0018] Further, the multi-parameter measurement device comprises a second light beam emitting device, a second lens group, a first reflecting mirror, a polarization beam splitter prism and a quarter-wave plate, a position sensitive detection system and a phase sensitive detection system.

[0019] Further, the second light beam emitting device is a continuous wave single frequency laser with a wavelength different from the working wavelength of the first light beam emitting device.

[0020] Further, the position sensitive detection system comprises a D-shaped mirror and a balanced detector.

[0021] Further, the phase sensitive detection system comprises a second reflecting mirror, a first lens, a spatial modulation grating and a phase shift detection device.

[0022] Further, the instantaneous velocity power spectral density is fitted using the following formula:

[0023]

[0024] In the formula, S v (w) is the power spectral density of the velocity, η is the fluid viscosity, T is the temperature, k is the optical trap stiffness, w is the frequency, R is the radius of the microparticle, k B is the Boltzmann constant, R is the thermodynamic temperature, ρ f is the solution density, ρ p is the density of the cell to be measured.

[0025] Compared with the prior art, the beneficial effects of the present application are as follows:

[0026] The present application utilizes an optical tweezer capturing device to capture cells, and through a multi-parameter measurement device, based on multiple interactions between a probe light source and the captured cells, wherein the backscattered and forward scattered probe light by the cells is used to: through a position sensitive detection system, the motion trajectory of the restricted Brownian motion of the measured cells is measured, and through a phase sensitive detection system, the spatial distribution of the phase delay of the contents of the measured cells to the probe light is obtained. The data measured by the multi-parameter measurement device multiple times is averaged and denoised, the cell trajectory obtained by the experiment is processed as the instantaneous velocity power spectral density, and the spatial distribution of the phase delay of the contents of the measured cells to the probe light obtained by the experiment is processed as the phase shift distribution of the probe light after penetrating the cells. Then through a calculation program, based on the theory of the restricted Brownian motion of particles considering the fluid memory effect, the water content of the cells is calculated by theoretically fitting the instantaneous velocity power spectral density; based on the linear relationship between the density of non-water contents and the refractive index increment, the dry mass of the cells is obtained by two-dimensional integration within the cell profile. The method can measure the dry mass and water content of living cells simultaneously under the premise of non-contact and without changing the environment of the cells, and the optical equipment of the method is compatible with optical microscopes and other devices. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A structure diagram of a cell mass measurement device provided for embodiment 1 of the present application is provided.

[0028] Figure 2 A structure diagram of a sample cell in the cell mass measurement device provided for embodiment 1 of the present application is provided.

[0029] Figure 3 A graph of the instantaneous velocity power spectral density of the measured cells provided for embodiment 1 of the present application is provided. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the present application will be described more fully below. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0031] Embodiment 1

[0032] The present embodiment provides a method for simultaneously measuring the water content and dry mass of living cells, which comprises the following steps:

[0033] (1) Referring to Figure 1 , a living cell mass measurement device based on optical tweezers is built, and the measurement device comprises an optical tweezer capturing device and a multi-parameter measurement device;

[0034] The optical tweezer capturing device comprises a first light beam emitting device 1, a first lens group 2, a first dichroic mirror 3, a first objective lens 4, a sample cell 5, a second objective lens 6, and a second dichroic mirror 7, wherein,

[0035] The first light beam emitting device 1 adopts a continuous single-frequency laser with a center wavelength of 473 nm; the first dichroic mirror 3 and the second dichroic mirror 7 are selected to be dichroic mirrors with high reflectivity for a wave band near 473 nm and high transmissivity for other wave bands; the first objective lens 4 is selected to be an objective lens with a high magnification of 100x and a numerical aperture of 1.2 to focus the continuous single-frequency Gaussian light beam with a center wavelength of 473 nm as much as possible; the second objective lens 6 is selected to be an objective lens with a magnification of 10x; the sample cell 5 is composed of a glass slide 5.1, a cover glass 5.2, and a 170-micron-thick double-sided fixed adhesive 5.3, and the thickness of the cover glass 5.2 is 0.17 mm; the sample cell 5 contains a solution 5.5, and the cells to be measured 5.4 exist in the solution 5.5 in the sample cell 5, and the whole sample cell 5 is clamped on a three-dimensional motorized translation stage 5.6;

[0036] The multi-parameter measurement device comprises a second light beam emitting device 12, a second lens group 13, a first reflecting mirror 14, a polarization beam splitter prism 9 and a quarter-wave plate 8, a position-sensitive detection system, and a phase-sensitive detection system, wherein,

[0037] The second light beam emitting device 12 adopts a continuous single-frequency laser with a center wavelength of 671 nm;

[0038] The position-sensitive detection system comprises a D-shaped mirror 10 and a balanced detector 11; the D-shaped mirror 10 needs to have a sharp edge to divide the light beam as evenly as possible and reduce light loss, and the balanced detector 11 is selected to be a high-speed balanced homodyne detector;

[0039] The phase-sensitive detection system comprises a second reflecting mirror 15, a first lens 16, a spatial modulation grating 17, and a phase shift detection device 18; the phase shift detection device 18 adopts a commercial CCD (charge coupled device) camera, which can image the illumination wave band of the continuous single-frequency laser with a center wavelength of 671 nm;

[0040] (2) The cells to be measured 5.4 are captured by the optical tweezer capturing device, so that they are bound in the optical trap and do restricted Brownian motion:

[0041] The 473nm light beam emitted by the first light beam emitting device 1 is expanded by the first lens group 2, and the expanded light beam is reflected by the first dichroic mirror 3 and then enters the first objective lens 4 to form a strongly focused Gaussian light beam; the strongly focused Gaussian light beam enters the sample cell 5 to make the cell trapped in the center of the optical trap to do the confined Brownian motion, and to find a suitable cell 5.4 to be measured; the three-dimensional electric translation stage 5.6 is used to move the cell 5.4 to be measured in the sample cell 5 to the vicinity of the optical trap until the cell 5.4 to be measured is trapped by the optical tweezer; the Gaussian light beam is reflected out of the light path by the second dichroic mirror 7 after being converged by the second objective lens 6, and the trapping of the cell to be measured is completed.

[0042] (3) The multi-parameter measurement device illuminates the cell to be measured based on one probe light source, so that the probe light is divided into backscattered light and forward scattered light by the cell to be measured:

[0043] The 671nm light beam emitted by the second light beam emitting device 12 is expanded by the second lens group 13, and the expanded light beam is reflected by the first mirror 14 and then passes through the polarization beam splitter prism 9 to become linearly polarized light, and then passes through the quarter-wave plate 8 to rotate the polarization plane by 45° and then passes through the second dichroic mirror 7 to enter the second objective lens 6, and the second objective lens 6 converges the reflected light beam to illuminate the sample cell 5, and the illumination light generates backscattered light and forward scattered light after passing through the cell 5.4 to be measured;

[0044] (4) The backscattered light is used to measure the motion trajectory of the cell to be measured doing the confined Brownian motion at a preset measurement time resolution; and the forward scattered light is used to generate the spatial distribution of the phase delay of the cell content to the probe light:

[0045] The backscattered light collected by the second objective lens 6 passes through the transmission of the second dichroic mirror 7 and then enters the quarter-wave plate 8, and the linearly polarized light deflected by 45° by the quarter-wave plate 8 is rotated by 45° again, and compared with the linearly polarized light passing through the polarization beam splitter prism 9, the polarization plane is rotated by 90°, so the backscattered light is reflected by the polarization beam splitter prism 9 to the position sensitive detection system, and the backscattered light is divided into two beams by the D-shaped mirror 10 and carries the position information of the cell 5.4 to be measured to enter two balanced detectors 11, and the balanced detectors 11 convert the light intensity signals into voltage signals A and B, and then U0 is calculated by subtracting the two signals and dividing the sum of the two signals, and then the motion trajectory of the cell 5.4 to be measured is derived;

[0046] The forward scattered light collected by the first objective lens 4 passes through the transmission of the first dichroic mirror 3 and then passes through the second mirror 15 and the first lens 16 to enter the phase sensitive detection system, and the spatial modulation grating 17 is placed a short distance before the phase shift detection device 18 to copy the incident wavefront, and the spatial modulation grating 17 is used to record the spatial distribution of the phase delay of the cell content 5.4 to the probe light in the process of small-range propagation and the light itself.

[0047] (5) The motion trajectory data of multiple measurements are collected for averaging and denoising, and the processed trajectory of the measured cell is subjected to fast Fourier transform to obtain the instantaneous velocity power spectral density, as shown in Figure 3 The spatial distribution of the phase delay of the probe light is processed in real time, and the phase shift change data of each point is extracted to obtain the phase shift distribution after the probe light transmits through the cell.

[0048] (6) The instantaneous velocity power spectral density is fitted by the restricted Brownian motion theory of the fluid memory effect of the microparticles, and the fitting formula is as follows:

[0049]

[0050] In the formula, S v (w) is the power spectral density of the velocity, η is the fluid viscosity, T is the temperature, k is the optical trap stiffness, w is the frequency, R is the radius of the microparticles, k B is the Boltzmann constant, R is the thermodynamic temperature, ρ f is the density of the solution, and ρ p is the density of the measured cell. By fitting the instantaneous velocity power spectral density, ρ p and R can be obtained.

[0051] The following formula is used to calculate the water content of the cell:

[0052]

[0053] In the formula, m * is the water content of the measured cell, R is the radius of the microparticles, ρ f is the density of the solution, and ρ p is the density of the measured cell.

[0054] Based on the linear relationship between the non-water content and the refractive index increment, the dry mass of the cell is obtained by two-dimensional integration within the cell profile, and the calculation formula is as follows:

[0055]

[0056] In the formula, m is the dry mass of the measured cell, is the phase change value, α is the specific refractive increment, k0 is the wave vector, and σ is the cell plane.

[0057] According to the calculation, the water content of the measured cell 5.4 is 7.2×10 -11 g, and the dry mass is 2.2×10 -11 g.

[0058] Compared with other existing measurement methods, the present application not only realizes the synchronous measurement of dry mass and water content of living cells, but also has fast measurement speed, can rapidly sample displacement information in a short time (tens of milliseconds), and does not need to process complex images.

[0059] The above-described embodiments only express specific examples of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A method for synchronously measuring the water content and dry mass of living cells, characterized in that: The method is implemented using an optical tweezers capture device and a multi-parameter measurement device; the method comprises the following steps: (1) The cell to be tested is captured by an optical tweezers capture device, so that it is confined in an optical trap and performs restricted Brownian motion; (2) A multi-parameter measurement device is used to illuminate the cells to be measured based on a probe light source, so that the probe light is divided into backscattered light and forward scattered light by the cells to be measured; (3) Using backscattered light to measure the trajectory of the cell under test performing confined Brownian motion at a preset measurement time resolution; using forward scattered light to generate the spatial distribution of the phase delay of the cell contents to the probe light; (4) Perform fast Fourier transform on the trajectory of the cell to be tested to obtain the instantaneous velocity power spectrum density; process the spatial distribution of the phase delay of the probe light in real time, extract the phase shift change data of each point, and obtain the phase shift distribution after the probe light passes through the cell; (5) The instantaneous velocity power spectrum density is fitted by the restricted Brownian motion theory of particles with fluid memory effect to calculate the water content of the cell. Based on the linear relationship between the density of non-aqueous content and the refractive index increment, the cell dry mass is obtained by two-dimensional integration within the cell contour. The instantaneous velocity power spectral density is fitted using the following formula: ； Where, is the power spectral density of velocity, is the fluid viscosity, is the temperature, is the light trap stiffness, is the frequency, is the particle radius, is the Boltzmann constant, is the solution density, is the cell density to be measured; Use the following formula to solve for the water content of the cell: ； Where, is the water content of the cell to be tested, is the particle radius, is the solution density, is the cell density to be measured.

2. The method according to claim 1, characterized in that The optical tweezers capture device includes a first light beam emitting device, a first lens group, a first dichroic mirror, a first objective lens, a sample cell, a second objective lens, and a second dichroic mirror; the first light beam emitting device is a continuous wave single-frequency laser with a wavelength in the blue-green spectrum range of 400~600nm.

3. The method according to claim 1, characterized in that The multi-parameter measurement device includes a second light beam emitting device, a second lens group, a first reflecting mirror, a polarization beam splitter prism and a quarter wave plate, a position sensitive detection system and a phase sensitive detection system.

4. The method according to claim 3, characterized in that The second light beam emitting device is a continuous wave single frequency laser having a wavelength different from the operating wavelength of the first light beam emitting device.

5. The method according to claim 3, characterized in that The position sensitive detection system is a D-shaped mirror and a balanced detector.

6. The method according to claim 3, characterized in that The phase sensitive detection system includes a second reflector, a first lens, a spatial modulation grating and a phase shift detection device.

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