Live cell observation and drug stimulation experiment by inverted laser confocal microscope

Abstract

The application discloses an experimental method for live cell observation and drug stimulation by using an inverted laser confocal microscope, which comprises the following steps: placing a supporting frame with a hollow structure in a laser confocal small dish of the inverted laser confocal microscope, then placing a flaky carrier material loaded with live cells on the supporting frame in an inverted manner, and making the live cells on the flaky carrier material be located at the hollow structure of the supporting frame, so that a part of an observation surface of the flaky carrier material is kept in a natural suspended state; and then observing the live cells on the observation surface in the natural suspended state by using an objective lens of the inverted laser confocal microscope. By the method, pressure on the live cells can be avoided during the observation, and the cells can be stimulated by drugs, and the effect of the drugs on the cells can be observed in time, so that the application range of the inverted laser confocal microscope is expanded, and the utilization rate is improved.

Description

Technical Field

[0001] This invention belongs to the field of cell observation technology, specifically relating to an experimental method for observing live cells and applying drugs to stimulate them using an inverted laser confocal microscope. Background Technology

[0002] Laser confocal microscopy is a very common experimental instrument. It is a modern optical microscope that uses a laser as a light source, building upon traditional fluorescence microscopy. Through the use of laser scanning and conjugate focusing devices, and with the aid of a computer, it performs digital image processing on the observed object. It can acquire fluorescently labeled images of cells or tissues at extremely high resolution, observe the fine structure and morphological changes within cells or tissues, observe changes in the concentration of important intracellular ions or pH at the subcellular level, and observe and record cellular physiological activities using electrophysiological techniques. Using laser scanning confocal microscopy, it is also possible to perform tomographic imaging of observed samples, reconstruct and analyze the three-dimensional spatial structure of cells.

[0003] Based on their structure, laser confocal microscopes can be divided into upright laser confocal microscopes and inverted laser confocal microscopes. Inverted laser confocal microscopes have a wider range of applications in medical research, allowing observation of fixed tissues or subcellular structures, or observation of live cells cultured in petri dishes. They can meet the needs of observation under different magnifications (10X, 20X, 40X, 60X, 100X) or oil immersion lenses. Upright laser confocal microscopes have a water immersion lens, allowing observation of live cells by inserting the lens into the culture medium with the cell-bearing material upright. However, the magnification of the water immersion lens is limited to 40X and 60X. Both inverted and upright laser confocal microscopes are expensive, costing approximately 4 million yuan each. Therefore, due to budget constraints, medical schools are unlikely to equip their research facilities with both types of laser confocal microscopes. Because of their wider applications, inverted laser confocal microscopes are more commonly used, while upright laser confocal microscopes are relatively less common.

[0004] However, existing techniques for observing live cells on material surfaces using inverted laser confocal microscopy involve directly inverting the cell-bearing material into the laser confocal dish. This exerts pressure on the cells, potentially affecting the experimental results. Furthermore, if drug stimulation is required during observation, the gap between the cells and the dish bottom is too small, preventing the drug from reaching the cells and ultimately hindering its effectiveness. While using an upright laser confocal microscope can alleviate this situation to some extent, the high cost of both types of microscopes limits their availability. Therefore, expanding the applicability of inverted laser confocal microscopy to enable real-time observation and drug stimulation of live cells on material surfaces has become particularly urgent. Summary of the Invention

[0005] The purpose of this invention is to provide an experimental method for observing live cells and applying drugs using an inverted laser confocal microscope, so as to avoid stressing the live cells during the observation process, apply drugs to stimulate the cells, and observe the effect of drugs on the cells in a timely manner, thereby expanding the applicability of the inverted laser confocal microscope and improving its utilization rate.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides an experimental method for observing live cells and applying drugs using an inverted laser confocal microscope, comprising:

[0008] Place a hollow support frame in the laser confocal dish of the inverted laser confocal microscope, then invert the sheet-like carrier material carrying live cells onto the support frame, and position the live cells on the sheet-like carrier material at the hollow structure of the support frame, so that part of the sheet-like carrier material to be observed is in a natural suspended state.

[0009] Then, the objective lens of an inverted laser confocal microscope is used to observe the living cells on the surface to be observed, which are in a naturally suspended state.

[0010] This invention provides an experimental method for live cell observation and drug stimulation using an inverted laser confocal microscope. First, a support frame is placed between the inverted sheet-like carrier material seeded with live cells and the bottom of the laser confocal microscope dish. This allows the live cells on the sheet-like carrier material to be positioned within the hollow structure of the support frame, maintaining a portion of the observed surface of the sheet-like carrier material in a naturally suspended state. This avoids pressure on the cells and prevents interference with experimental results, thus solving the problems of existing technologies. Simultaneously, the support frame creates a gap between the sheet-like carrier material and the bottom of the laser confocal microscope dish, allowing drugs added to the dish to act on the cells seeded on the surface of the sheet-like carrier material. This solves the problem in existing technologies where drugs are difficult to apply to cells on the carrier material surface.

[0011] Furthermore, in a preferred embodiment of the present invention, the shape of the support frame is cross-shaped, circular, or herringbone-shaped.

[0012] Preferably, the support frame is shaped like a cross. This support frame minimizes obstruction of the carrier material surface, thus avoiding excessive obstruction of the living cells on the carrier material surface. Furthermore, because it divides the carrier material into four quadrants with four openings, it facilitates drug delivery through all four openings, thereby effectively stimulating the cells.

[0013] Preferably, in a preferred embodiment of the present invention, the support frame is made of pure metal.

[0014] Preferably, in a preferred embodiment of the present invention, the support frame is a pure titanium metal cross. This cross has the advantages of being resistant to deformation, sterilized, and reusable, making it very environmentally friendly. Furthermore, titanium metal has good biocompatibility and no toxic side effects on cells.

[0015] Preferably, in a preferred embodiment of the present invention, the thickness of the support frame is 0.3 to 0.8 mm.

[0016] Preferably, in a preferred embodiment of the present invention, the thickness of the support frame is 0.45 mm or 0.65 mm.

[0017] Furthermore, in a preferred embodiment of the present invention, the objective lens has a magnification of 20x or 40x.

[0018] Preferably, in a preferred embodiment of the present invention, the support frame is a pure titanium metal cross with a thickness of 0.45 mm, and the objective lens is a 40x laser confocal microscope;

[0019] Alternatively, the support frame is a pure titanium metal cross with a thickness of 0.65mm, and the objective lens is a 20x laser confocal microscope.

[0020] Preferably, in a preferred embodiment of the present invention, the laser confocal dish is of type 0.

[0021] Although the support frame is used to pad the carrier material, providing sufficient space for the drug to effectively act on living cells, in order to focus under 40x and 20x objectives, the laser confocal dish used must first be "size 0", which has the thinnest bottom. Secondly, if focusing at 40x and ensuring sufficient space for the drug to act on the cells, the thickness of the pure titanium cross should be 0.45mm. If focusing at 20x and ensuring sufficient space for the drug to act on the cells, the thickness of the pure titanium cross can be 0.65mm.

[0022] Furthermore, in a preferred embodiment of the present invention, the sheet-like carrier material is a circular pure titanium sheet. Preferably, in a preferred embodiment of the present invention, the surface morphology of the circular pure titanium sheet is 5V nanotubes with a diameter of 10 mm and a thickness of 1 mm.

[0023] These nanotubes are prepared by anodizing at 5V, followed by cell seeding on their surface. Of course, other materials or materials with different morphologies can also be used, not limited to pure titanium discs and nanotubes. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a circular pure titanium sheet used in an experimental example of the present invention.

[0025] Figure 2 This is a schematic diagram of the pure titanium cross in the experimental example of the present invention;

[0026] Figure 3 This is a schematic diagram of the laser confocal dish "0" in the experimental example of this invention;

[0027] Figure 4 This is a schematic diagram of placing a pure titanium cross into a laser confocal dish numbered "0" in an experimental example of the present invention;

[0028] Figure 5 This is a schematic diagram of placing a circular pure titanium sheet upside down on a pure titanium cross in an experimental example of the present invention;

[0029] Figure 6 These are images of the experimental group in the experimental examples of this invention, focused under a 0.65mm titanium support and a 20x objective lens;

[0030] Figure 7 The image shows the control group in the experimental example of this invention focused under a 0.65mm titanium support and a 20x objective lens;

[0031] Figure 8These are images of the experimental group in the experimental examples of this invention, focused under a 0.45mm titanium support and a 40x objective lens;

[0032] Figure 9 The image shows the control group in the experimental example of this invention focused under a 0.65mm titanium support and a 20x objective lens;

[0033] Figure 10 This is a focused image of the experimental group after drug administration in the experimental examples of this invention, under a 20x objective lens;

[0034] Figure 11 This is a focused image of the experimental group after drug administration in the experimental examples of this invention, taken under a 40x objective lens.

[0035] Figure 12 This is a focused image of the control group (without medication) in the experimental examples of this invention, taken under a 20x objective lens.

[0036] Figure 13 This is a focused image of the control group (without medication) in the experimental examples of this invention, taken under a 40x objective lens. Detailed Implementation

[0037] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] Example 1

[0039] This embodiment provides an experimental method for observing live cells and applying drugs using an inverted laser confocal microscope, including:

[0040] A hollow pure titanium metal cross (0.45 mm thick) is placed in the laser confocal dish of an inverted laser confocal microscope. Then, a circular pure titanium metal sheet (5V nanotube surface morphology, 10 mm in diameter and 1 mm thick) carrying live cells is placed upside down on the pure titanium metal cross, with the live cells on the circular pure titanium metal sheet located in the hollow structure of the pure titanium metal cross, so that part of the circular pure titanium metal sheet to be observed is in a natural suspended state.

[0041] The living cells on the surface to be observed, which are in a naturally suspended state, are then observed using a 40x objective lens of an inverted laser confocal microscope.

[0042] Example 2

[0043] This embodiment provides an experimental method for observing live cells and applying drugs using an inverted laser confocal microscope, including:

[0044] A hollow pure titanium metal cross (0.65 mm thick) is placed in the laser confocal dish of an inverted laser confocal microscope. Then, a circular pure titanium metal sheet (5V nanotube surface morphology, 10 mm in diameter and 1 mm thick) carrying live cells is placed upside down on the pure titanium metal cross, with the live cells on the circular pure titanium metal sheet located in the hollow structure of the pure titanium metal cross, so that part of the circular pure titanium metal sheet to be observed is in a natural suspended state.

[0045] The living cells on the surface to be observed, which are in a naturally suspended state, are then observed using a 20x objective lens of an inverted laser confocal microscope.

[0046] Example 3

[0047] This embodiment provides an experimental method for observing live cells and applying drugs using an inverted laser confocal microscope, including:

[0048] A hollow pure titanium herringbone support (0.45 mm thick) is placed in the laser confocal dish of an inverted laser confocal microscope. A circular pure titanium sheet (5V nanotube surface morphology, 10 mm in diameter, and 1 mm thick) containing live cells is then placed upside down on the pure titanium herringbone support, with the live cells on the circular pure titanium sheet located in the hollow structure of the pure titanium herringbone support, so that part of the circular pure titanium sheet to be observed is in a natural suspended state.

[0049] Then, the living cells on the surface to be observed, which is in a naturally suspended state, are observed using a 40x objective lens of an inverted laser confocal microscope.

[0050] Experimental Example

[0051] To verify the effectiveness of the method for observing live cells on the surface of a carrier material and simultaneously performing drug treatment under inverted laser confocal microscopy, the following experiment was conducted:

[0052] Experiment Title: Recording of cytoplasmic calcium ion oscillations in mouse bone marrow mesenchymal stem cells (Mus BMSCs) on the surface of titanium nanotubes and observation of calcium pool-manipulated calcium ion influx.

[0053] Experimental observation indicators:

[0054] ① 0.65mm pure titanium cross + 5V titanium nanotube pure titanium disc seeded with Mus BMSCs + inverted laser confocal 20x objective lens: Can it focus? Do the cells respond after drug addition?

[0055] ② 0.45mm pure titanium cross + 5V titanium nanotube pure titanium metal disc seeded with Mus BMSCs + inverted laser confocal 40x objective lens: whether it can focus, whether the cells react after drug addition.

[0056] Experimental materials:

[0057] An alcohol lamp; eight circular pure titanium metal sheets with a surface morphology of 5V nanotubes, each 10 mm in diameter and 1 mm thick (e.g., ...). Figure 1 (As shown); forceps; one 24-well plate; complete cell culture medium (79% α-MEM (gibco); fetal bovine serum (incellgene); 1% penicillin antibody (incellgene)); BMSCs in logarithmic growth phase that have reached 80% of the area of ​​a 100mm dish; PBS buffer (incellgene); EDTA trypsin (incellgene); cell culture incubator (Thermo); 15ml centrifuge tubes (AuGene TECH); centrifuge (Cence. Xiangyi L500); cell counter (CellDrop BF, DeNOVIX); four pure titanium crosses with thicknesses of 0.65mm and 0.45mm (e.g.) Figure 2 As shown, its "cross" arm is 19mm long; HBSS (buffered saline, Solarbio); FLUO-8 (a calcium ion green fluorescent dye, abcam); "0" laser confocal dish (e.g., Figure 3 As shown, Cellvis (D29-20-0-N); calcium-free buffer (140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM glucose (all from Sinopharm Reagent), 10mM HEPES, 20μM EGTA (Aladdin)); probenecid (a reagent that enhances the staining effect of FLUO-8 on cells, G-CLONE); inverted laser confocal microscope (Olympus, FV1000); TG (carotene, an endoplasmic reticulum stress inducer, MCE).

[0058] Experimental steps:

[0059] ① Experimental Groups:

[0060]

[0061]

[0062] 40x objective lens group: 40x objective lens for inverted laser confocal microscope + 0.45mm pure titanium cross;

[0063] 20x objective lens group: 20x objective lens for inverted laser confocal microscope + 0.65mm pure titanium cross;

[0064] Experimental group: group receiving drug stimulation; Control group: group receiving no drug stimulation.

[0065] ② Heat the back of the 5V nanotube titanium sheet with an alcohol lamp and let it cool.

[0066] ③ Label the 24-well cell culture plate, place the cooled titanium sheet into the 24-well plate, and add 1 ml of total culture medium to each well.

[0067] ④ Take 80% density BMSCs in a 100mm dish and in the logarithmic growth phase, wash with 3ml PBS, digest with 5ml EDTA in a 37℃ cell incubator for 5min, stop digestion with 5ml of complete culture medium, and pipette the cells.

[0068] ⑤ After pipetting, transfer to a 15ml centrifuge tube and centrifuge at 800r / min for 5min.

[0069] ⑥ Remove the supernatant, resuspend in 2 ml of whole culture medium, and mix thoroughly by pipetting.

[0070] ⑦ Measure and calculate the cell density, then seed 3*10⁵ cells per well into the wells containing titanium sheets, shake well, and incubate at 37°C for 24 hours.

[0071] ⑧ Sterilize four titanium crosses (0.45mm and 0.65mm) that have been soaked in 75% alcohol overnight with high temperature using an alcohol lamp, and then let them cool.

[0072] ⑨ BMSCs cultured for 24 h were rinsed with 1 ml of PBS per well and aspirated.

[0073] ⑩ Add 1 ml of HBSS and 10 μM of FLUO-8 to each well and incubate at 37°C for 30 min.

[0074] The laser confocal dish is labeled "0", and a pure titanium cross is placed inside (e.g., Figure 4 As shown in the image, add 1 ml of calcium-free buffer solution to each small dish.

[0075] After incubating the BMSCs with FLUO-8, rinse them once with HBSS without aspirating them, and transfer the titanium plate into a small dish, placing it upright.

[0076] Add 2 mM probenecid to each small dish and incubate at 37°C for 30 min.

[0077] After probenecid incubation is complete, gently pick up the titanium sheet with tweezers and place it upside down on the titanium cross (e.g., ...). Figure 5 (As shown).

[0078] After removing the cap from the laser confocal dish, place it on an inverted laser confocal microscope and observe it under a laser with an excitation wavelength of 488 nm and an emission wavelength of 520-570 nm. Focus under a 0.65 mm titanium support and a 20x objective lens. The experimental group (e.g.) is visible. Figure 6 As shown), the control group (as shown) Figure 7 All (as shown) can be clearly focused; when focusing under a 0.45mm titanium support + 40x objective lens, the experimental group (such as...) is clearly visible. Figure 8 As shown), the control group (as shown) Figure 9 All (as shown) can be clearly focused.

[0079] After 1 minute, TG at a concentration of 2.5 μM was added to the laser confocal dish in the 40x and 20x objective lens groups, while no TG was added to the control group. It can be seen that at 1 minute, the 20x objective lens group (e.g., Figure 10 The experimental groups (as shown) and the 40x objective lens group (as shown) Figure 11 The green fluorescence of the control group (as shown in the image) was significantly enhanced, while that of the control group under 20x objective lens (as shown in the image) was significantly enhanced. Figure 12 (as shown) and the 40x objective control group (as shown) Figure 13 (As shown) Without the addition of TG, the fluorescence intensity did not change. (This indicates that BMSCs on titanium nanotubes supported by either 0.45 mm or 0.65 mm pure titanium scaffolds responded to TG stimulation, demonstrating that the two thicknesses of the cross structure allow the cells to respond effectively to drug stimulation.)

[0080] Experimental results:

[0081] ① 0.65mm pure titanium cross + 5V titanium nanotube pure titanium disc seeded with Mus BMSCs + inverted laser confocal 20x objective lens: can focus, cells show obvious response after drug addition.

[0082] ② 0.45mm pure titanium cross + 5V titanium nanotube pure titanium disc seeded with Mus BMSCs + inverted laser confocal 40x objective lens: can focus, cells show obvious response after drug addition.

[0083] Experimental conclusion:

[0084] After the material is supported by a pure titanium cross under inverted laser confocal microscopy, live cells on its surface can be observed and drug treatment can be performed simultaneously.

[0085] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for observing live cells using an inverted laser confocal microscope for non-diagnostic purposes, characterized in that, It includes: A hollow support frame is placed in the laser confocal dish of an inverted laser confocal microscope. Then, a sheet-like carrier material carrying live cells is placed upside down on the support frame, with the live cells on the sheet-like carrier material located in the hollow structure of the support frame, so that part of the surface of the sheet-like carrier material to be observed is in a naturally suspended state. Then, the live cells on the surface to be observed, which are in a naturally suspended state, are observed using the objective lens of an inverted laser confocal microscope. The support frame is a pure titanium metal cross with a thickness of 0.45mm, and the objective lens is a 40x laser confocal microscope; Alternatively, the support frame is a pure titanium metal cross with a thickness of 0.65 mm, and the objective lens is a 20x laser confocal microscope; The laser confocal dish is model number 0; The sheet-like carrier material is a circular pure titanium sheet; The surface morphology of the circular pure titanium sheet is 5V nanotubes, with a diameter of 10 mm and a thickness of 1 mm.

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