CN112501016B

CN112501016B - Laser confocal microscope carrier liquid device for eukaryotic cell culture

Info

Publication number: CN112501016B
Application number: CN202011207536.2A
Authority: CN
Inventors: 侯森; 阮奕铭; 钟正; 吕冰; 戴洪萍; 王文瑜
Original assignee: Shandong Huapai Testing Technology Co ltd; Jinan University
Current assignee: Shandong Huapai Testing Technology Co ltd; Jinan University
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2023-08-01
Anticipated expiration: 2040-11-02
Also published as: CN112501016A

Abstract

The invention provides a laser confocal microscope carrier liquid device for eukaryotic cell culture, which comprises a lower carrier, an upper carrier, a sealing gasket, a bolt, a glass slide and a separated upper cover, wherein the upper carrier is arranged on the lower carrier; the surface center department of carrier has offered the big round hole that runs through down, the surface left and right sides symmetry of carrier has been offered down and has been run through the round hole, carrier top is equipped with the slide glass down, the slide glass lid closes big round hole top in the middle of the carrier down, slide glass top is equipped with sealed the pad, sealed pad top is equipped with the carrier, the big round hole top lid of going up the carrier is equipped with disconnect-type upper cover, two bolts pass in proper order down the carrier with sealed the pad, and fix in the round hole of going up the carrier, the slide glass is by the fixation clamp down between the carrier with the sealed pad. The invention has the advantages of convenient disassembly, repeated use and reduced environmental treatment pressure and use cost, and can enter a sterilizing pot for sterilizing operation.

Description

Laser confocal microscope carrier liquid device for eukaryotic cell culture

Technical Field

The invention relates to the technical field of experimental devices, in particular to a laser confocal microscope carrier liquid device for eukaryotic cell culture.

Background

Confocal laser microscopy was first used by m.minsky at university of harvard in 1955. After decades of improvement, confocal laser microscopy has become an important tool for scientific research. Compared with a common microscope, the confocal laser microscope can inhibit a blurred image, obtain the optimal resolution without changing an objective lens, and further realize three-dimensional reconstruction of an optical slice. Local light operation can be performed, and information acquisition and construction can be performed on various samples marked by fluorescent dyes. Compared with a common microscope, the method has the advantages of accurate positioning and high scanning speed. Confocal laser microscopy is important in biological, environmental, metrology, medical, and materials research. The laser confocal microscope is widely applied to the fields of biological research for detecting molecules and structures in tissues and cells, measuring the change of ion concentration in the cells, realizing the measurement of fluorescence related data, observing the migration and growth of the cells for a long time, measuring the three-dimensional surface and researching the imaging of a spectrum. However, laser confocal microscopy uses pinhole imaging and inverted viewing, requiring the use of extremely thin glass sheets (typically cover slips 0.13-0.17mm thick) as the sample carrier device. The direct use of a slide and coverslip combination is difficult to operate and eukaryotic cells cannot be directly cultured. The cell culture dish used by the laser confocal microscope in the current market is high in price, cannot be sterilized at high temperature and can only be used once, and a special bracket is required to be configured to be fixed above the objective lens. In view of the fact that no mature laser confocal microscope carrier liquid device for eukaryotic cell culture exists at present, the invention provides a laser confocal microscope cell culture carrier liquid device which is low in price, can be used for repeated sterilization and is suitable for a common microscope bracket (such as a glass slide bracket), and the invention has high urgency and market value.

The laser confocal microscope is based on the principle of a traditional optical microscope, a laser scanning device is additionally arranged, a pinhole is used for blocking light rays outside a focal length so as to remove light rays outside the focus of a detector, and raster scanning is used for imaging. In actual use, the dye in the sample is excited by the laser, it is focused by a dichroic mirror onto a pinhole (pinhole), and the light passing through the pinhole is measured by a photomultiplier tube. The confocal laser microscope structure comprises a light source, a condenser, a detector, a light transmission device and the like. The laser confocal microscope is used for carrying out fluorescent marking on a study object, adopting a conjugate focusing mode to scan and observe a sample point by point to obtain a high-resolution confocal image, and realizing three-dimensional imaging by controlling a Z-axis focal plane.

Since confocal laser microscopes use pinhole imaging and inverted viewing, the distance between the observer and the lens is required to be very short, and therefore there is a high thickness requirement (a tolerable maximum thickness of about 0.17 mm) for the slide of the object. For such severe observation requirements, there are currently several solutions that can achieve the object of observation to some extent, but all have certain limitations. The first approach is to achieve sample observation by using a cover slip of a conventional microscope in combination with a slide. The method comprises the steps of firstly, adding a sample to be detected onto a glass slide, then covering a cover glass above the sample, further turning over the whole combination of the glass slide and the cover glass, and finally, placing a device obtained by turning over above an objective lens for observation. In the process, the sample to be measured is stirred due to too fast overturning, and overflows due to too slow overturning, so that observation is influenced. Meanwhile, because the space between the cover glass and the glass slide is small, a large sample cannot be placed, and cell culture cannot be performed. In addition, if the overflowed sample to be tested has toxicity, the sample to be tested has potential health risks for experimental staff. Although the cell culture dish of the common laser confocal microscope on the market can achieve the purpose of cell culture and does not need to be turned over during observation, the cell culture dish still has certain limitations. The culture dish consists of a culture dish cover, a culture dish body with holes and a cover glass. The bottom of the dish was glued with a cover glass of approximately 0.17mm thickness using a special bio-glue. The shape of such dishes requires a special objective stage device, which does not match the usual slide stage device. And this dish is only disposable, resulting in its use being expensive (about 10 RMB-well). If a laser confocal microscope carrier liquid device which can be sterilized repeatedly and is low in cost and is oriented to eukaryotic cell culture can be prepared, a great pushing effect can be generated on popularization of a laser confocal microscope and related instruments (such as a laser confocal fluorescence related spectrometer invented on the basis).

Aiming at the short plate of the common observation scheme of the current laser confocal microscope, a laser confocal microscope liquid-carrying device capable of being repeatedly sterilized is researched and developed, and experiments such as tabletting, disinfection, cell culture, imaging and the like are carried out on the device.

Disclosure of Invention

The invention aims to provide a reusable laser confocal microscope carrier liquid device which can be used for eukaryotic cell culture and can be used for a common glass slide objective table, and the device is used for solving the problems in the background technology.

The technical scheme adopted for solving the technical problems is as follows:

a laser confocal microscope carrier liquid device for eukaryotic cell culture is characterized in that: comprises a lower carrier, an upper carrier, a sealing gasket, a bolt, a glass slide and a separated upper cover; the center of the surface of the lower carrier is provided with a large through hole, the left side and the right side of the surface of the lower carrier are symmetrically provided with small through holes, the top of the lower carrier is provided with a glass slide, the glass slide covers the top of the large through hole in the middle of the lower carrier, the top of the glass slide is provided with a sealing pad, the center of the surface of the sealing pad is provided with a large through hole, the left side and the right side of the surface of the sealing pad at the same position with the small through holes on the two sides of the lower carrier are symmetrically provided with small through holes, the top of the sealing pad is provided with an upper carrier, the surface center department of going up the carrier has offered the big round hole that runs through, with the little round hole co-located of sealed pad both sides go up carrier left and right sides symmetry and seted up the little round hole that runs through, be equipped with in the little round hole with bolt assorted screw thread, the big round hole top lid of going up the carrier is equipped with the disconnect-type upper cover, two bolts pass in proper order the lower carrier with sealed pad, and fix in the little round hole of going up the carrier, the slide glass is by the fixation clamp under with between the sealed pad.

The upper carrier, the sealing gasket, the glass slide and the lower carrier can be limited and fixed through the bolts, the situation that the glass slide is broken is not easy to occur, and the bolts can rapidly detach the device.

Further, the sealing gasket is made of silica gel.

The sealing gasket covered on the glass slide is used for attaching and sealing the glass slide and the upper carrier, so that liquid leakage is avoided.

Further, the upper carrier and the lower carrier are made of transparent glass, and the transparent glass is high-strength high-temperature-resistant and high-pressure-resistant glass.

The uploading device and the downloading device are made of high-strength glass materials, and are not easy to bend and deform in the use process.

The high-strength glass material can bear high temperature and high pressure, can enter a sterilizing pot for sterilizing operation, and is repeatedly used, so that the environmental treatment pressure is reduced, and the use cost is reduced.

Further, the large round hole passes through the center of the round hole to break the lower carrier into two sections.

Further, the length of the sealing gasket is 76mm, the width is 26mm, and the thickness is 0.1mm.

Further, the slide glass has a length of 22mm, a width of 22mm, and a thickness of 0.17mm.

Further, the lower carrier has a length of 76mm, a width of 26mm, and a thickness of 2mm.

Further, the length of the upper carrier is 76mm, the width is 26mm, and the thickness is 10mm.

Further, the diameter of the large round hole is 20mm, and the diameter of the small round hole is 10mm.

Further, the length of the separated upper cover is 50mm, the width is 24mm, and the thickness is 0.17mm.

Compared with the prior art, the invention has the following beneficial effects:

1. eukaryotic cell culture can be performed and can be used on a conventional microscope stage.

2. The uploading device and the downloading device are made of glass materials with high strength, and are not easy to bend and deform in the using process.

3. The upper carrier, the sealing gasket, the glass slide and the lower carrier are limited and fixed through the bolt screws, so that the glass slide is not easy to crack.

4. The sealing gasket covered on the glass slide is used for attaching and sealing the glass slide and the upper carrier, so that liquid leakage is avoided.

5. The glass materials used by the uploading device and the downloading device can bear high temperature and high pressure, the device is convenient to detach, and the glass materials can enter a sterilizing pot to be sterilized after being detached and are repeatedly used, so that the environment treatment pressure is reduced, and the use cost is reduced.

Drawings

FIG. 1 is an overall front view of the present invention;

FIG. 2 is an overall top view of the present invention;

FIG. 3 is an overall side view of the present invention;

FIG. 4 is a 3D exploded view of the present invention;

FIG. 5 (a) is an image of the present invention before entering a sterilization kettle for high temperature and high pressure sterilization;

FIG. 5 (b) is an image of the present invention after being sterilized by a sterilization pot at high temperature and high pressure;

FIG. 5 (c) is an image of the invention after loading of the cell suspension by autoclaving in a sterilization kettle;

FIG. 6 (a) is a microscopic image of cells of the present invention cultured for 0 h;

FIG. 6 (b) is a microscopic image of cells of the present invention cultured for 12 hours;

FIG. 6 (c) is a microscopic image of cells of the present invention cultured for 24 hours;

FIG. 6 (d) is a microscopic image of cells of the present invention cultured for 36 hours;

FIG. 7 is an image under a fluorescence microscope after staining cells with Hoechst stain;

FIG. 8 is an image under a fluorescence microscope after staining cells with PI staining agent;

FIG. 9 (a) is a blue fluorescent cell image obtained by observing stained HeLa cells using a 20-fold objective lens of a confocal laser microscope;

FIG. 9 (b) is a red fluorescent cell image obtained by observing stained HeLa cells using a 20-fold objective lens of a confocal laser microscope;

FIG. 9 (c) is a red and blue fluorescence synthesized cell image obtained by observing the stained HeLa cells using a 20-fold objective lens of a confocal laser microscope;

FIG. 10 (a) is a blue fluorescent cell image obtained by observing stained HeLa cells using a 40-fold objective lens of a confocal laser microscope;

FIG. 10 (b) is a red fluorescent cell image obtained by observing the stained HeLa cells using a 40-fold objective lens of a confocal laser microscope;

FIG. 10 (c) is a red and blue fluorescence synthesized cell image obtained by observing the stained HeLa cells using a 40-fold objective lens of a confocal laser microscope;

wherein, 1-loader, 2-loader, 3-sealing pad, 4-bolt, 5-glass slide,

6-separating upper cover, 7-big round hole and 8-small round hole.

Detailed Description

The following describes specific embodiments of the invention with reference to the drawings and examples:

it should be noted that the structures, proportions, sizes, etc. shown in the drawings are merely for the purpose of understanding and reading the disclosure, and are not intended to limit the scope of the invention, which is defined by the appended claims.

Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.

Referring to fig. 1-4, fig. 1-4 depict the overall structure of the present invention, including a lower carrier 1, an upper carrier 2, a gasket 3, bolts 4, a slide 5, and a breakaway cover 6; the utility model discloses a glass slide, including carrier 1, upper carrier 2, lower carrier 1, slide 5, sealing pad 3, sealing pad 2's big round hole 7 top cover is equipped with big round hole 7 that runs through, with the little round hole 8 of lower carrier 1 same position department's of surface center department sealing pad 3's surface left and right sides symmetry has been seted up the little round hole 8 that runs through, sealing pad 3 top is equipped with carrier 2, go up carrier 2's surface center department has been seted up the big round hole 7 that runs through, with sealing pad 3 both sides little round hole 8 same position department go up carrier 2 left and right sides symmetry has been seted up the little round hole 8 that runs through, be equipped with in the little round hole 8 with bolt 4 assorted screw thread, the big round hole 7 top cover of upper carrier 2 is equipped with separation upper cover 6, two 4 pass in proper order under with sealing pad 3 and sealing pad 3 the carrier 2 is in the sealing pad 2, the carrier is fixed to be in between the carrier 1 and the glass slide 2.

Specifically, the gasket 3 is made of silica gel.

Specifically, the materials of the upper carrier 2 and the lower carrier 1 are transparent glass.

Specifically, the large round hole 7 cuts the lower carrier 1 into two parts through the center of the round hole.

Specifically, the length of the gasket 3 is 76mm, the width is 26mm, and the thickness is 0.1mm.

Specifically, the slide glass 5 has a length of 22mm, a width of 22mm, and a thickness of 0.17mm.

Specifically, the length of the lower carrier 1 is 76mm, the width is 26mm, and the thickness is 2mm.

Specifically, the length of the upper carrier 2 is 76mm, the width is 26mm, and the thickness is 10mm.

Specifically, the diameter of the large round hole 7 is 20mm, and the diameter of the small round hole 8 is 10mm.

Specifically, the length of the split type upper cover 6 is 50mm, the width is 24mm, and the thickness is 0.17mm.

Experiment 1: the feasibility of the invention for high-temperature high-pressure sterilization can be verified

In order to verify the feasibility of the invention to withstand high temperature and high pressure sterilization, the invention was sterilized at high temperature and high pressure (121 ℃,0.1mpa,30 minutes) using a sterilization pot in this experiment, and changes of the invention before and after sterilization were observed. Fig. 5 (a) shows an image before the invention enters a sterilizing pot to be sterilized at high temperature and high pressure, wherein the lower carrier 1, the upper carrier 2, the glass slide 5, the screw 4 and the screw thread 6 are not deformed and broken before sterilization. Fig. 5 (b) shows an image of the invention after high temperature and high pressure sterilization in a sterilization pot, wherein the downloader 1, the uploading device 2, the glass slide 5, the screw 4 and the screw 6 are all free from deformation and fragmentation after sterilization, and the whole invention is free from obvious change. Therefore, the invention can bear the high-temperature and high-pressure sterilization of the sterilizing pot. FIG. 5 (c) is a photograph of the invention after loading the cell suspension by high temperature and high pressure sterilization in a sterilizer, the invention can perfectly carry the cell suspension without leakage, and the separated upper cover can further prevent dust fall and excessive liquid evaporation during cell culture.

Experiment 2: verification of feasibility of observing cells cultured in the present invention using a microscope

To verify the feasibility of the invention to culture and observe cells using a microscope, the experiment was performed by culturing Hela cells in 90% DMEM high sugar medium supplemented with 10% Fetal Bovine Serum (FBS), 1% diantigen (100U/ml penicillin, 100ug/ml streptomycin), placing the cell suspension in a cavity surrounded by the carrier and slide glass of the liquid carrier device of the invention after autoclaving, covering the separated upper cover to prevent evaporation of water, placing at 37℃and 5% CO ₂ Cells were observed with a microscope after culturing for 0h, 12h, 24h and 36h, respectively, in humidified incubators, see fig. 6 (a), 6 (b), 6 (c) and 6 (d). FIG. 6 (a) shows that the cells cultured for 0h according to the present invention were not completely settled on the glass slide, and that some cells were suspended in the liquid medium, using a developing deviceThe micro mirror can clearly see that the cells settled on the glass slide are round, and the density is low; FIG. 6 (b) shows that cells cultured for 12 hours have settled on a glass slide for adherent growth, and that normal adherent development of Hela cells takes the shape of a shuttle; FIG. 6 (c) shows the gradual proliferation of cells cultured for 24 hours in the present invention, and a significant increase in cell density was observed; FIG. 6 (d) shows that the cells cultured for 36 hours in the present invention were further proliferated, and that the cell density was further increased, and the cells were gradually filled with a glass slide, and the development and proliferation conditions were good.

Thus, the cells can be cultured by adding the liquid culture medium in the invention, the growth state is normal, and the cells cultured in the device can be observed in real time by using a microscope, and the cells can be cultured while being observed, so that a cell image is obtained.

Experiment 3: verification of the feasibility of Using fluorescence microscopy to observe stained cells cultured in the present invention

The product can be used for not only laser confocal microscope observation but also common microscope (including fluorescence microscope) observation. To verify the feasibility of using a fluorescence microscope to observe the stained cells in the present invention, the experiment stained cultured Hela cells with Hoechst stain (1 mg/ml) and Propidium Iodide (PI) stain (1 mg/ml). Chromatin can shrink as cells undergo apoptosis. Hoechst 33342 can penetrate cell membranes, and the fluorescence of the apoptosis cells after staining can be obviously enhanced compared with that of normal cells. Propidium Iodide (PI) cannot penetrate cell membranes and cannot stain normal or apoptotic cells with intact cell membranes. Whereas for necrotic cells, the integrity of their cell membranes is lost, propidium Iodide (PI) can stain necrotic cells. After staining, the cells were observed using a fluorescence microscope, and the whole cell nuclei after staining the cells using Hoechst stain were blue-colored, see fig. 7, and fig. 7 is an image under a fluorescence microscope after staining the cells using Hoechst stain, wherein blue marks the cell nuclei of all cells. Dead nuclei after staining the cells with PI stain appear red, see figure 8. FIG. 8 is an image under a fluorescence microscope after staining cells with PI staining agent, wherein the nuclei of dead cells are red-labeled.

The above data indicate that cells cultured in the present invention can be stained with a stain and observed with a fluorescence microscope to obtain a stained cell image.

Experiment 4: verification of the feasibility of Using a confocal laser microscope to observe cells cultured in the present invention

In order to obtain a high-resolution observation image, when a laser confocal microscope is used for observing cells, the thickness of a glass slide is generally required to be 0.13-0.17mm, and the thickness of the glass slide used in the invention is 0.17mm, so that the requirement of the laser confocal microscope on the thickness of the glass slide is met. To verify the feasibility of using a laser confocal microscope to observe cells cultured in the present invention, the present experiment was carried out by culturing Hela cells in 90% DMEM high-sugar medium supplemented with 10% Fetal Bovine Serum (FBS), 1% diantigen (100U/ml penicillin, 100ug/ml streptomycin), and in the device of the present invention sterilized at high temperature, and placing at 37℃5% CO ₂ Culturing for 12h in a humidified incubator, after the cells are completely settled on a glass slide and attached to the glass slide, treating the cells by using hydrogen peroxide, and then dyeing the Hela cells by using Hoechst 33342 and a PI (polyimide) coloring agent. After the dyeing is finished, observing the cells by using a 20-time objective lens of a laser confocal microscope, and obtaining dyed cell images, wherein the dyed cell images are shown in fig. 9 (a), 9 (b) and 9 (c), and all Hela cell nuclei of the cells dyed by the Hoechst dye in fig. 9 (a) are blue, have a large number and are uniformly distributed in a visual field; FIG. 9 (b) shows the red color and a small number of dead nuclei of Hela stained with PI stain; fig. 9 (c) shows a red-blue field image obtained by overlapping a red-field image and a blue-field image.

Observing the same group of stained Hela cells by using a 40-fold objective lens (oil lens) of a laser confocal microscope, wherein the obtained cell images are shown in fig. 10 (a), 10 (b) and 10 (c), and fig. 10 (a) shows that all Hela cell nuclei stained by Hoechst stain are blue, and compared with the number of cell nuclei observed in a 20-fold objective lens field of view, the number of cell nuclei in the 40-fold objective lens field of view is smaller, and single cells are larger; FIG. 10 (b) shows the red color of the nuclei of Hela dead cells stained with PI stain; fig. 10 (c) shows a red-blue field image obtained by overlapping a red-field image and a blue-field image.

The data show that the ordinary objective lens and the oil lens under different multiplying powers of the confocal laser microscope can be used for observing the cells which are cultured in situ and are dyed in the invention, and clear blue and red fluorescent cell images can be obtained.

The invention provides a laser confocal microscope carrier liquid device which is low in price, can be used for repeated sterilization and is applicable to eukaryotic cell culture of a common microscope bracket (such as a glass slide bracket). Compared with the defects of incapability of repeated use, high cost, incapability of culturing cells and the like of the traditional confocal laser microscope culture dish, the invention has the characteristics of high temperature and high pressure resistance, firm and durable material, repeated use, cell culture, different multiplying powers of various microscopes, observation by different kinds of objective lenses (common objective lenses and oil lenses) and the like, and well solves the problems. The invention fixes the common cover glass through the device to form the liquid-carrying cell culture observing device, can be easily obtained in each laboratory, only needs to replace the cover glass each time, and has high practicability.

While the preferred embodiments of the present invention have been described in detail with reference to the drawings, the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Many other changes and modifications may be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the specific embodiments, but only by the scope of the appended claims.

Claims

1. A laser confocal microscope carrier liquid device for eukaryotic cell culture is characterized in that: comprises a lower carrier (1), an upper carrier (2), a sealing gasket (3), a bolt (4), a glass slide (5) and a separated upper cover (6); the center of the surface of the lower carrier (1) is provided with a large through hole (7), the left and right sides of the surface of the lower carrier (1) are symmetrically provided with small through holes (8), the top of the lower carrier (1) is provided with a glass slide (5), the glass slide (5) is covered on the top of the large through hole (7) in the middle of the lower carrier (1), the top of the glass slide (5) is provided with a sealing gasket (3), the center of the surface of the sealing gasket (3) is provided with the large through hole (7), the left and right sides of the surface of the sealing gasket (3) at the same position as the small through holes (8) at the two sides of the lower carrier (1) are symmetrically provided with small through holes (8), the top of the sealing gasket (3) is provided with an upper carrier (2), the length of the upper carrier (2) is 76mm, the width is 26mm, the center of the surface of the upper carrier (2) is provided with the large through hole (7), the same position as the small through holes (8) at the two sides of the sealing gasket (3) is provided with the small through holes (8), the top of the upper carrier (2) is provided with the small through holes (6) is provided with the same length as the small through holes (8) and the small through holes (6) are separated from the top of the upper carrier (2), the width is 24mm, and thickness is 0.17mm, and two bolts (4) pass in proper order carrier (1) down with sealed pad (3), the length of carrier (1) down is 76mm, and width is 26mm, and thickness is 2mm, and fix in small round hole (8) of carrier (2) down, slide glass (5) are by the fixation clamp carrier (1) down with sealed pad (3) between, the length of slide glass (5) is 22mm, and width is 22mm, thickness is 0.17mm.

2. The eukaryotic cell culture laser confocal microscope carrier liquid device according to claim 1, wherein the sealing gasket (3) is made of silica gel.

3. The eukaryotic cell culture laser confocal microscope carrier liquid device according to claim 1, wherein the upper carrier (2) and the lower carrier (1) are made of transparent glass, and the transparent glass is high-strength high-temperature-resistant and high-pressure-resistant glass.

4. The eukaryotic cell culture's confocal laser microscope carrier liquid device according to claim 1, characterized in that the big round hole (7) crosses its centre of a circle to break the lower carrier (1) into two sections.

5. The eukaryotic cell culture confocal microscope carrier liquid device according to claim 2, characterized in that the sealing gasket (3) has a length of 76mm, a width of 26mm and a thickness of 0.1mm.