A biological sample preparation device and a biological sample preparation method

Abstract

The present invention provides a biological sample preparation device, comprising a sample tank that can be placed in a liquid environment, a glass slide placed in the sample tank for carrying biological samples, a transparent film covering the sample tank with the same refractive index as the liquid environment, and a sample pressing ring tightly fastened to the sample tank to fix the transparent film on the sample tank; the bottom of the sample tank is a hollow light-transmitting design, and the transparent film isolates the biological sample from the liquid environment. The present invention also provides a method for sample preparation using the biological sample preparation device. The biological sample preparation device and sample preparation method provided by the present invention can isolate biological samples from liquid environments, can be widely used in upright microscopes, inverted microscopes, or upright and inverted combined microscopes, and can image samples over a long period of time.

Description

Technical Field

[0001] The invention relates to the technical field of sample preparation devices, and in particular to a biological sample preparation device and a sample preparation method thereof. Background Art

[0002] Upright microscopes are widely used because of their advantages such as shorter optical path, easier to find key areas and better imaging effects. However, upright microscopes with high numerical aperture often require the use of liquid media for imaging. For example, light sheet upright microscopes are widely used in real-time high temporal and spatial resolution imaging studies of small model organisms due to their fast and low bleaching characteristics. However, since most of them use multiple water immersion objectives for imaging, the sample needs to be immersed in a liquid environment for imaging. Figure 1 When observing biological samples with a high numerical aperture upright microscope, the biological samples remain active in a liquid environment, and the objective lens of the upright microscope is immersed in the liquid environment to image the biological samples.

[0003] However, since many biological samples cannot be imaged under liquid conditions, this greatly limits the scope of application of upright microscopes. Take nematodes as an example. Nematodes are about 1 mm long and are characterized by a translucent body. The body of the nematode swings violently in a liquid environment, making it impossible to collect images. Currently, biological samples are usually fixed in gel to isolate the liquid and then imaged with an upright microscope. However, the gel dissolving temperature is relatively high, reaching the stress temperature of many samples such as nematodes. Therefore, high-resolution upright microscopes cannot be used in many samples. Currently, inverted microscopes, such as laser confocal microscopes, are used to image nematodes, but laser confocal microscopes are increasingly unable to meet research needs in terms of time and spatial resolution.

[0004] If the nematodes can be isolated from the liquid environment without changing the refractive index from the liquid to the nematodes, then an upright microscope can be used to collect data faster and conduct more in-depth studies on the nematodes at the subcellular level, which also expands the application range of upright microscopes such as light sheet microscopes. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a biological sample preparation device and a sample preparation method thereof which can isolate a biological sample from a liquid environment.

[0006] In order to solve the above technical problems, the present invention provides a biological sample preparation device, including a sample tank that can be placed in a liquid environment, a glass slide placed in the sample tank for carrying a biological sample, a transparent film covering the sample tank with the same refractive index as the liquid environment, and a sample pressing ring tightly fastened to the sample tank to fix the transparent film on the sample tank; the bottom of the sample tank is hollow and light-transmitting, and the transparent film isolates the biological sample from the liquid environment.

[0007] Furthermore, a layer of light-transmissive sample survival medium is arranged between the glass slide and the biological sample, the thickness of the sample survival medium is equal to the distance from the upper surface of the glass slide to the top of the sample groove, and the transparent film covering the sample groove can fix the biological sample on the sample survival medium.

[0008] Furthermore, the sample pressing ring is fixed in the sample slot, the glass slide in the sample slot is fixed in the sample slot, and the biological sample on the glass slide is immersed in the liquid environment.

[0009] Furthermore, the thickness of the film is 0.025 mm to 0.5 mm.

[0010] Furthermore, the sample slot is circular, square or other shapes, and the shape and size of the glass slide match the sample slot to ensure that the sample slot can hold the glass slide.

[0011] Furthermore, the sample slot is a sample slot with an arm or a sample slot without an arm.

[0012] Furthermore, the sample tank is made of pure titanium, titanium alloy or stainless steel.

[0013] The present invention also provides a method for preparing samples using the biological sample preparation device, comprising the following steps:

[0014] Place the slide in the sample well;

[0015] The sample survival medium is arranged above the glass slide so that a sample survival medium plane is formed in the sample groove;

[0016] Place the sample on a sample survival medium plane;

[0017] Cover the sample tank with a transparent film and press the sample onto the sample survival medium plane in the sample tank;

[0018] Fasten the sample pressing ring onto the sample slot to fix the transparent film onto the sample slot;

[0019] Place the sample chamber on an upright microscope for observation and imaging.

[0020] The present invention provides a biological sample preparation device and a sample preparation method thereof, which seals the biological sample in a sample slot with a thin film having the same refractive index as that of the ambient liquid, thereby isolating the biological sample from the external liquid environment, not only does it not affect the observation and imaging of the biological sample by a light sheet type upright microscope, but also does not cause the biological sample to be contaminated or deformed, and can be stored for a long time and reused multiple times, while also avoiding the phase difference caused by the change in refractive index. In addition, the present invention provides a biological sample preparation device, which places the biological sample on a glass slide in the sample slot, and can place a large number of biological samples at the same time, thereby allowing rapid high-throughput screening and improving the extraction efficiency. Moreover, the material selection and geometric dimensions of the sample slot of the present invention are designed for microscopic observation, and can be applied to most optical microscope observations, expanding the scope of use of the sample preparation device, and also expanding the scope of use of biological samples for observation and imaging, such as the observation of nematodes under upright microscopes such as light sheets, promoting the study of nematodes at the subcellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the structure of using an objective lens of an upright microscope immersed in a liquid environment to image a biological sample in the prior art;

[0022] Figure 2 A schematic structural diagram of a biological sample preparation device provided by an embodiment of the present invention;

[0023] Figure 3 A schematic diagram of the structure of a biological sample preparation device using a sample slot with an arm provided in an embodiment of the present invention;

[0024] Figure 4 A schematic diagram of the structure of a biological sample preparation device for conventional sample preparation in a liquid environment is provided in another embodiment of the present invention. DETAILED DESCRIPTION

[0025] See also Figure 2, a biological sample preparation device provided by an embodiment of the present invention comprises a sample tank 1 that can be placed in a liquid environment, a glass slide 2 placed in the sample tank 1 for carrying biological samples, a transparent film 3 covering the sample tank 1 and having the same refractive index as the liquid environment, and a sample pressing ring 4 tightly buckled on the sample tank 1 to fix the transparent film 3 on the sample tank 1. Among them, the bottom of the sample tank 1 is hollowed out and can support a cover glass, so that light can pass through the hollowed-out part of the bottom of the sample tank 1 for observation under an upright microscope, and the non-hollowed-out part of the bottom of the sample tank 1 is used to carry the glass slide 2. The thickness of the transparent film 3 is 0.025mm to 0.5mm, and the inner diameter of the sample pressing ring 4 is slightly smaller than the sum of the outer diameter of the sample tank 1 and 2 times the thickness of the film. In addition, the side wall of the sample tank 1 is designed in a trapezoidal shape, so that when the sample pressing ring 4 is buckled on the sample tank 1, the friction force can increase as the sample pressing ring 4 is pressed down, which can play an anti-slip and fixing role, thereby ensuring that the sample pressing ring 4 presses the film 3 tightly on the sample tank 1. Thus, when the sample tank 1 is placed in a liquid environment and observed with an upright microscope, the transparent film 3 can isolate the biological sample in the sample tank 1 from the external liquid environment, and can also prevent the environmental liquid from penetrating into the sample tank 1. In order not to change the refractive index of the objective lens to the sample, the refractive index of the film 3 should be the same as the refractive index of the environmental liquid. For example, a film made of fluorinated ethylene propylene or other films with the same refractive index as the environmental liquid can be selected according to the environmental liquid. Of course, since the sample preparation device can isolate the sample from the liquid environment, it can be observed and imaged not only with an upright microscope, but also with an inverted microscope, and a microscope combining an upright and an inverted microscope.

[0026] The size and shape of the sample tank 1 can be selected according to the specific situation to meet the needs of different equipment. The shape of the sample tank 1 can be round, square or other shapes. The shape and size of the slide glass 2 match the shape and size of the sample tank 1 to ensure that the sample tank 1 can hold the slide glass 2. The sample tank 1 is made of pure titanium, titanium alloy or stainless steel. The sample tank 1 made of these materials is not easy to deform and has good chemical stability and high and low temperature tolerance, and can be recycled. The assembled sample tank 1 can be disassembled and cleaned as a whole, or it can be placed in ultraviolet irradiation or a strong acid environment for cleaning. After cleaning and drying, the sample tank 1 can be stably stored at room temperature, and is not prone to oxidation and rust.

[0027] Among them, sample slot 1 is a sample slot without an arm, see Figure 3The sample tank 1 can also be made into a sample tank with an arm, the sample arm 6 is fixedly connected to the sample tank 1 by bolts, and the end of the sample arm 6 can be fixed to the sample operation table by screws, so that during the operation, only the sample arm 6 needs to be fixed to observe and image the biological sample in the sample tank 1. Since the size of the sample tank 1 can be designed in various specifications according to the size of the slide 2, and the sample tank 1 is made of a material that is not easy to deform and has good chemical stability and high and low temperature tolerance, it is designed to facilitate microscopic observation. Therefore, the sample prepared by the sample preparation device provided by the present invention can be used for most optical microscope observations, and users without relevant experience can also use it directly.

[0028] During the test, in order to ensure the activity of the biological sample, a layer of sample survival medium 5 can usually be set on the slide 2, and then the biological sample can be placed on the sample survival medium 5, so that it is convenient to observe the biological sample for a long time. The thickness of the sample survival medium 5 should be equal to the distance from the upper surface of the slide 2 to the top of the sample tank 1. After the transparent film 3 is covered on the sample tank 1 and fastened on the sample tank 1 with the sample pressing ring 4, the sample tank 1 is immersed in a liquid environment. The biological sample is isolated from the liquid environment in the sample tank 1, and the film 3 provides downward pressure to the biological sample under the force of the sample pressing ring 4, which can fix the biological sample on the sample survival medium 5 and ensure that the biological sample is in a stable state. As a specific embodiment of the present invention, if the biological sample is nematodes, zebrafish or fruit flies, the sample survival medium 5 is selected from solid agarose gel that can ensure the activity of nematodes, zebrafish or fruit flies. Of course, if other biological samples are selected, the sample survival medium 5 is selected from other media that can ensure the activity of the biological sample.

[0029] The sample preparation method of the biological sample preparation device provided by the present invention comprises the following steps:

[0030] Place the slide glass 2 in the sample slot 1;

[0031] The sample survival medium 5 is arranged above the glass slide 2 so that a sample survival medium plane is formed in the sample tank 1;

[0032] Place the sample on a sample survival medium plane;

[0033] Cover the sample slot 1 with a transparent film 3, and press the sample onto the sample survival medium plane in the sample slot 1;

[0034] Fasten the sample pressing ring 4 onto the sample slot 1 to fix the transparent film 3 onto the sample slot 1;

[0035] In this way, the sample tank 1 can be placed in a liquid environment and observed and imaged using an upright microscope.

[0036] The following uses the preparation of nematode samples as an example to specifically illustrate the method for using a biological sample preparation device provided in an embodiment of the present invention.

[0037] 1. Required Materials

[0038] Sample tank 1; glass slide 2; equal water refractive index film 3; nematode sample; sample pressing ring 4; sample survival medium 5, i.e., agarose gel; sterile deionized water; anesthetic, etc.

[0039] 2. Experimental Preparation

[0040] 1) Adjust the temperature to 20℃ and start the equipment for precooling;

[0041] 2) ultrasonically cleaning the glass slide 2;

[0042] 3) Prepare agarose gel at a ratio of 3% (W / V):

[0043] 3. Sample preparation process. Since nematodes' bodies swing violently or cannot survive in a liquid environment, a device that isolates the sample from the liquid environment is required when making nematode samples.

[0044] 1) embedding the glass slide 2 into the sample slot 1;

[0045] 2) Liquid agarose gel 5 is dripped onto the glass slide 2 in the sample tank 1;

[0046] 3) Cover the gel with a coverslip and flatten it to squeeze out the excess gel;

[0047] 4) After forming, the cover glass is removed, and a gel plane is formed above the sample tank 1, and the excess solid gel outside the sample tank is removed;

[0048] 5) Use a pipette to absorb and drop 1-3 μL of nematode anesthetic onto the gel surface, and then pick nematodes at different developmental stages and add them to the anesthetic;

[0049] 6) Before the anesthetic dries, place the nematodes in the desired direction and then cover them with a transparent film 3 having the same refractive index as water;

[0050] 7) The sample pressing ring 4 is buckled on the film and pressed down, and the film 3 is between the sample pressing ring and the sample slot. The friction force ensures that the sample pressing ring is not easy to fall off. At the same time, the film 3 presses the nematode on the sample survival medium 5 in the sample slot 1 to ensure the survival of the sample. At the same time, the pressure on the nematode further limits the swing of the nematode;

[0051] 8) The model can be immersed in a liquid environment and isolated from the liquid without changing the refractive index from the objective lens to the sample, and then observed and imaged with an upright microscope.

[0052] The order of the device from top to bottom during the specific assembly process is: sample pressing ring 4, equal water refractive index film 3, biological sample, sample survival medium 5, slide 2 and sample tank 1. The entire device isolates the liquid environment, and the transparent film 3 on the sample surface provides downward pressure, providing double protection for stabilizing the sample. It is worth mentioning that since the slide 2 and sample survival medium 5 at the bottom of the sample are both light-transmissive, the sample can also be imaged from the bottom using an inverted microscope at the same time, and the sample preparation device provided by the present invention can achieve multifunctional applications.

[0053] Of course, as another embodiment, the biological sample preparation device provided by the present invention can also be used for conventional sample preparation in a liquid environment. Figure 4 For biological samples that will not sway violently in a liquid environment and will not cause the inability to collect images, the biological samples can be attached to a glass slide 2, and then the glass slide 2 attached to the biological sample can be placed at the bottom of the sample tank 1. A sample pressing ring 4 with an outer diameter slightly smaller than the inner diameter of the sample tank 1 can be used to fix the sample tank 1 in the sample tank 1, and the glass slide 2 at the bottom of the sample tank 1 can be fixed. Finally, the sample tank 1 is placed in a liquid environment, and an upright microscope is used for observation and imaging. The order from bottom to top of the sample tank 1 placed in the liquid environment is: sample tank 1-slide 2-sample-sample pressing ring 4.

[0054] The following uses cell sample preparation as an example to specifically describe a method for using a biological sample preparation device provided by another embodiment of the present invention. Cells need to remain alive in a liquid environment, so when preparing cell samples, there is no need to use a device that isolates the sample from the liquid environment, and the cells can be directly immersed in the liquid environment for observation.

[0055] 1. Required Materials

[0056] The sample preparation device; sample tank 1; glass slide 2; cell culture medium; sample pressing ring 4; cells and cell culture conditions;

[0057] 2. Experimental Preparation

[0058] 1) Turn on the equipment and preheat it to 37°C. Set the carbon dioxide in the living cell workstation to 5%;

[0059] 2) Ultrasonic cleaning of slide 2

[0060] 3. Sample preparation:

[0061] 1) Spread the cells onto slide 2 and place them in a 37°C, 5% carbon dioxide incubator overnight; the next day, transfect the cells with plasmids;

[0062] 2) 16 hours after transfection, the glass slide 2 is taken out and placed in the sample slot 1 of the sample preparation device.

[0063] 3) Place a sample pressing ring 4 whose outer diameter is close to the inner diameter of the sample tank 1 onto the glass slide 2 in the sample tank 1 and press the glass slide 2 tightly, relying on the friction between the sample pressing ring 4 and the inner wall of the sample tank 1 to keep the sample pressing ring 4 stable;

[0064] 4) Fill the sample tank 1 with culture medium to maintain the activity of the cell sample;

[0065] 5) Blow dry the bottom of the slide 2 and place it on an inverted microscope for imaging.

[0066] 4. Storage and use

[0067] After using the sample preparation device, wipe it clean with alcohol, dry it and store it at room temperature. It can be used indefinitely.

[0068] The biological sample preparation device provided by the present invention expands the application scope of upright microscopes such as light sheet microscopes, and also develops the use of non-liquid environment imaging samples under upright microscopes. The device can be applied to both upright and inverted microscopes. Compared with gel embedding sample preparation, the transparent film with the same refractive index as the environmental liquid is used to avoid the phase difference caused by the change of refractive index when observing the imaging. The sample preparation device can place multiple samples at a time, thereby efficiently screening positive clones, and avoiding sample stress caused by high gel embedding temperature. At the same time, the device is easy to operate and can be recycled.

[0069] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A biological sample preparation device, characterized in that: The invention comprises a sample tank (1) which can be placed in a liquid environment, a glass slide (2) placed in the sample tank (1) for carrying a biological sample, a transparent film (3) covering the sample tank (1) and having the same refractive index as the liquid environment, and a sample pressing ring (4) which is tightly fastened to the sample tank (1) and fixes the transparent film (3) on the sample tank (1); the bottom of the sample tank (1) is of a hollow light-transmitting design, and the transparent film (3) isolates the biological sample from the liquid environment; A layer of light-transmissive sample survival medium (5) is arranged between the glass slide (2) and the biological sample. The thickness of the sample survival medium (5) is equal to the distance from the upper surface of the glass slide (2) to the top of the sample groove (1). The transparent film (3) covering the sample groove (1) can fix the biological sample on the sample survival medium (5).

2. The biological sample preparation device according to claim 1, characterized in that: The sample pressing ring (4) is fixed in the sample groove (1), and the glass slide (2) in the sample groove (1) is fixed in the sample groove (1), and the biological sample on the glass slide (2) is immersed in the liquid environment.

3. The biological sample preparation device according to claim 1, characterized in that: The thickness of the film is 0.025 mm to 0.5 mm.

4. The biological sample preparation device according to claim 1, characterized in that: The shape of the sample slot (1) is circular, square or other shapes, and the shape and size of the glass slide (2) match those of the sample slot (1) to ensure that the sample slot (1) can accommodate the glass slide (2).

5. The biological sample preparation device according to claim 1, characterized in that: The sample trough (1) is a sample trough with an arm or a sample trough without an arm.

6. The biological sample preparation device according to claim 1, characterized in that: The sample tank (1) is made of pure titanium, titanium alloy or stainless steel.

7. The sample preparation method of the biological sample preparation device according to any one of claims 1 to 6, characterized in that: The steps include: placing a glass slide (2) in a sample chamber (1); The sample survival medium (5) is arranged above the glass slide (2) so that a sample survival medium plane is formed in the sample groove (1); Place the sample on a sample survival medium plane; Covering the sample tank (1) with a transparent film (3) and pressing the sample onto the sample survival medium plane in the sample tank (1); Fasten the sample pressing ring (4) onto the sample slot (1) to fix the transparent film (3) onto the sample slot (1); The sample tank (1) is placed on an upright microscope for observation and imaging.

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