Sample container with FEP film for carrying biological samples for optical microscopic observation

Abstract

In optical microscopy including fluorescence microscopy, a biological sample (215) can be immersed in an aqueous liquid medium (210) during observation. Due to the refractive index mismatch between the liquid medium and a conventional glass bottom container holding the sample, image resolution is reduced when viewing the sample. To improve resolution, a sample container (100) is formed with a container body (110) having a bottom (120) with an aperture (125), a fluorinated ethylene propylene copolymer (FEP) film (150) attached to the bottom (120) to cover the aperture (125) to form a cavity-facing surface (155) on the film (150), and exposed to the liquid medium (210). Since the refractive index of FEP is close to that of water, image resolution is improved. Furthermore, the cavity-facing surface (155) is activated by plasma treatment to promote adhesion of the cavity-facing surface (155) to the biological sample (215) to enable cells in the biological sample (215) to proliferate in adherent cell culture and to immobilize the biological sample (215) during observation.

Description

Cross-reference to related applications

[0001] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 492,800, filed March 29, 2023, the disclosure of which is incorporated herein by reference in its entirety. List of abbreviations

[0002] 3D

[0003] ABS Acrylonitrile Butadiene Styrene

[0004] Argon gas

[0005] FEP (Fluorinated Ethylene Propylene Copolymer)

[0006] MTF Modulation Transfer Function

[0007] PLA (polylactic acid)

[0008] UV Technical Field

[0009] This invention relates to a sample container for holding biological samples immersed in an aqueous liquid medium, which, compared to conventional glass-bottom containers, offers the advantage of improved image resolution of the biological sample during optical microscopy examination; the invention also relates to an optical microscopy system using the sample container. Background Technology

[0010] In this specification and the appended claims, the refractive index of material s is expressed as n. s .

[0011] The term "aqueous liquid medium" as used in this article refers to a liquid containing water as its main component or solvent. Please note that the refractive index of an aqueous liquid medium is primarily determined by water.

[0012] Cell biology, or cell science, deals with the structure, function, proliferation, pathology, and life cycle of cells. Cell studies are conducted using microscopy, cell culture, and cell fractionation. Adherent cell culture is commonly used in cell science research. In adherent cell culture, cells grow in a nutrient solution and attach to the surface of a cell culture vessel to form a cultured cell line. Cell biologists can then observe the cultured cell line under a microscope. Subcellular structures, such as organelles and specific regions within the cell that perform particular tasks, are often objects of interest for microscopic examination of cultured cell lines. Due to the small size of subcellular structures, imaging individual cells within a cultured cell line typically requires high image resolution.

[0013] When observing biological samples containing cultured cell lines under an optical microscope, a sample container is used to hold the biological sample within the microscope, with the sample immersed in a nutrient solution, typically an aqueous liquid medium. Consider the case where the optical microscope is an inverted microscope, observing the biological sample from the bottom of the sample container. The resolution of the image of the biological sample observed under an optical microscope is determined by the MTF of the optical microscope system, which includes the biological sample (which is the image source), the aqueous liquid medium (in which the biological sample is immersed), the sample container, and the optical microscope. Typically, glass is the preferred material for holding biological samples immersed in an aqueous liquid medium, and the sample container is typically, or preferably, a conventional glass-bottomed container. However, the refractive index of glass (typically 1.500–1.690; hereinafter referred to as n) is limited. 玻璃 =1.52 (representative value) and the refractive index of water (n 水 The difference is significant (e.g., 1.333). As a result, image distortion occurs when images of biological samples are transferred from an aqueous liquid medium to a glass-bottomed container. This image distortion affects the modulation transfer function (MTF), leading to a reduction in image resolution.

[0014] A direct approach to minimize image resolution degradation is to treat the sample container as an optical lens and determine the geometric and optical details of the lens to optimize the MTF of the resulting optical microscope system for maximizing the resolution of the images captured by the optical microscope. However, this approach is not attractive because: (1) it is costly to manufacture sample containers with precise geometric and optical details as lenses; and (2) sample containers are typically single-use and not reused for different biological samples.

[0015] There is a need in the art for an improved sample container that, compared to conventional glass-bottomed containers, can improve image resolution when imaging biological samples immersed in an aqueous liquid medium using an optical microscope without significantly increasing manufacturing costs. Summary of the Invention

[0016] A first aspect of this disclosure aims to provide a sample container for holding biological samples for microscopic observation, wherein the biological sample is immersed in an aqueous liquid medium during microscopic observation. This sample container can improve image resolution during microscopic observation of biological samples while enabling the sample container to be manufactured at low cost.

[0017] The sample container comprises a container body and a membrane. The container body includes a bottom and a cavity located at the bottom. The cavity is used to contain a liquid medium and a biological sample. At least one pore is formed in the bottom. The membrane is attached to the bottom to completely cover the at least one pore, such that a cavity-facing surface is formed on the membrane, exposed to the cavity. The cavity-facing surface allows the liquid medium to directly contact the membrane, enabling the light beam forming the image of the biological sample to be transmitted from the liquid medium through the membrane to the microscope without passing through the bottom. Specifically, the membrane is an FEP membrane made of FEP, and the cavity-facing surface is an activated surface that enhances the adhesion of the cavity-facing surface to the biological sample. Advantageously, compared to conventional glass-bottom containers, this allows the biological sample to adhere to the cavity-facing surface, and improves the image resolution of the images acquired by the microscope due to the close refractive indices between FEP and water.

[0018] In addition to the cavity-facing surface, the thin film also includes an outward-facing surface that is opposite to the cavity-facing surface.

[0019] In some implementations, the container body can be made of any material.

[0020] In some implementations, the container body is made of glass, fused silica, or any 3D printing material.

[0021] In some implementations, the thickness of the film is between 1 nm and 5 mm (inclusive).

[0022] In some implementations, the cavity-facing surface is activated such that its adhesion to biological samples is at least comparable to that of a glass surface.

[0023] In some implementations, the cavity-facing surface is activated by plasma treatment. Generally, plasma treatment is performed in a gas-filled environment.

[0024] In some implementations, the gas is a rare gas selected from argon, helium, neon, krypton, xenon, and radon.

[0025] In some implementations, the purity level of the gas is less than or equal to 100%.

[0026] In some implementations, the gas flow rate during plasma treatment is between 0.001 L / min and 10 L / min (inclusive).

[0027] In some implementations, the gas pressure is up to 10 MPa during plasma treatment.

[0028] In some implementations, the gas is excited at a power between 1 mW and 5000 W (inclusive) during plasma treatment.

[0029] In some implementations, plasma treatment is performed over a duration of up to 24 hours.

[0030] In some implementations, the outward surface is the unactivated, pristine surface of the thin film.

[0031] Generally, the container body is a rigid body of any shape. In some embodiments, the container body has a circular or rectangular shape when viewed from above.

[0032] In some embodiments, at least one well consists of multiple independent wells, thereby allowing the sample container to additionally function as a multi-well cell culture plate for culturing multiple isolated biological samples prior to microscopic observation. Multiple isolated biological samples collectively constitute a biological sample.

[0033] A second aspect of this disclosure is to provide an optical microscope system.

[0034] The optical microscope system includes an optical microscope and a sample container for holding biological samples for observation by the optical microscope, according to any of the disclosed embodiments.

[0035] Optical microscope systems may also include a refractive matching solution / material that matches the refractive index of the thin film. Specifically, the refractive matching solution / material is filled between the sample container and the optical microscope to avoid additional distortion of the image when the image of the biological sample leaves the thin film through the outward surface.

[0036] In some implementations, the refractive matching solution / material is chosen to be water.

[0037] In some embodiments, the optical microscope is a fluorescence microscope that includes an excitation optics system and a detection optics system. The excitation optics system is configured to generate an excitation beam and use the generated excitation beam to illuminate the biological sample, thereby exciting the biological sample to fluoresce, resulting in a fluorescence image of the biological sample. The detection optics system is configured to capture and amplify the fluorescence image, thereby forming a microscope image of the biological sample.

[0038] In some implementations, the excitation beam is a UV beam.

[0039] In some implementations, the fluorescence microscope is a confocal microscope.

[0040] In some embodiments, the fluorescence microscope is a light-sheet microscope, and the resulting excitation beam is an excitation light sheet. If UV light is used as the excitation beam, then the excitation light sheet is a UV light sheet.

[0041] In some implementations, the fluorescence microscope is also an upright microscope system or an inverted microscope system.

[0042] In some implementations, the fluorescence microscope is also a tilting microscope system.

[0043] In some implementations, the excitation optics and the detection optics are controllably moved relative to the sample container, thereby exciting different layers of the biological sample sequentially by the excitation light sheet, and then imaging is performed by the detection optics to obtain the three-dimensional structure of the biological sample.

[0044] Other aspects of this disclosure are disclosed as shown in the following embodiments. Attached Figure Description

[0045] Figure 1 A perspective view and a cross-sectional view of a sample container according to an exemplary embodiment of the present disclosure are depicted.

[0046] Figure 2 Depicting the use Figure 1 An optical microscope system for a sample container, wherein the sample container operates under typical working conditions, wherein the sample container contains a biological sample immersed in an aqueous liquid medium, and the biological sample is being observed through a microscope.

[0047] Figure 3 A variation of the sample container is depicted, which has multiple independent pores at the bottom instead of a single pore. This allows the sample container to be used additionally as a multi-well cell culture plate for culturing multiple isolated biological samples prior to microscopic observation.

[0048] Figure 4 It shows Figure 2 The optical microscope system is a fluorescence microscope system with a fluorescence microscope, wherein the fluorescence microscope system is also a tilting microscope system and the fluorescence microscope is a light sheet microscope.

[0049] Figure 5 The increased adhesion of plasma-activated FEP films to biological samples was demonstrated by depicting the culture results of CT26 cells cultured on (a) glass, (b) plasma-treated FEP films, and (c) untreated FEP films.

[0050] Those skilled in the art will understand that the elements in the figures are shown for simplicity and clarity and are not necessarily depicted to scale. Detailed Implementation

[0051] This disclosure provides a sample container for holding biological samples for microscopic observation while the biological samples are immersed in an aqueous liquid medium. The main advantage of this sample container compared to conventional glass-bottom containers is the improved resolution of the images of the biological samples observed under a microscope. Furthermore, the manufacturing cost of this sample container is significantly lower than the expensive option of redesigning the sample container to minimize the MTF of the final optical microscope system.

[0052] This disclosure also provides an optical microscope system for observing biological samples using the disclosed sample container.

[0053] Before detailing the implementation scheme of the disclosed sample container, the following is a summary of the reasoning used in developing the sample container.

[0054] In traditional glass-bottomed containers, the refractive index mismatch between the aqueous liquid medium and the glass bottom leads to reduced image resolution when imaging biological samples using a microscope. The refractive indices of water and glass are n and n, respectively. 水 =1.333 and n 玻璃 =1.52, therefore their refractive index ratio is n 水 / n 玻璃 =1.14. This indicates that considerable beam refraction occurs when the light beam travels from the first medium, water, to the second medium, glass. If the bottom of the glass is replaced with a bottom made of some material x, in n 水 =n x In this case, the light beam does not refract, thus avoiding image distortion during propagation and minimizing image resolution degradation. However, choosing a material x with the same refractive index as water is not always the optimal choice for maximizing image resolution, as the aqueous liquid medium into which biological samples are immersed is not pure water. The refractive indices of 10%, 20%, and 60% glucose aqueous solutions have been reported to be 1.3477, 1.3635, and 1.4349, respectively. Therefore, choosing a material x with a refractive index around 1.33 to 1.36 is reasonable.

[0055] When selecting material x, the first considerations are: (1) the refractive index of material x should be between 1.33 and 1.36. Other selection considerations are as follows, and material x should meet the following requirements: (2) it should be highly transparent to visible light; (3) it should be able to form a thin film with a surface for adhesion to biological cells and sufficient mechanical strength; (4) it should be impermeable to water; and (5) it should be non-toxic to biological cells. Since fluorescence microscopy is often combined with UV irradiation in cell studies to reveal the precise location, development, and function of tissues and cells, (6) material x is preferably a material that is highly transparent to UV light.

[0056] After extensive research, the inventors discovered that FEP is a suitable material as material x. The refractive index of FEP is n. FEP =1.344. Therefore, the ratio of the refractive indices of FEP and water is n. FEP / n 水 =1.008, which is closer to 1 than the corresponding ratio between glass and water (1.14). This demonstrates the advantage of using an FEP bottom container instead of a traditional glass bottom container in improving image resolution.

[0057] FEP is known to have high transparency and good transmittance to both visible and UV light. Films can be manufactured from FEP using conventional injection molding and screw extrusion techniques. FEP films can be manufactured to have the mechanical strength required for use as container bottoms. FEP films are known to be impermeable to water and non-toxic to biomaterials. However, the surface of FEP films is inert, making it difficult for biological cells to adhere to them. The inventors have discovered that activating the FEP film surface through plasma treatment significantly improves the adhesion of biological cells to the film surface. For information on FEP film activation, see [link to relevant documentation]. P.,L.Peterková,S.Rimpelová,A.Pinkner,N. Kasálková,Z.Kolská,T.Ruml,andV. "Plasma activated perfluoroethylenepropylene for cytocompatibility enhancement," Polymer Degradation and Stability, 130(2016):277-287, the entire contents of which are incorporated herein by reference.

[0058] The sample container disclosed in this article was developed based on the above observations and findings.

[0059] A first aspect of this disclosure is to provide a sample container for holding biological samples for microscopic observation. During microscopic observation, the biological sample is immersed in an aqueous liquid medium.

[0060] The following uses Figure 1 and Figure 2 Describe the sample container. Figure 1 A perspective view and a cross-sectional view of an exemplary sample container 100 are depicted. Figure 2A cross-sectional view of the sample container 100 is depicted, corresponding to a scenario in which the sample container 100 is in a typical working state: it contains a biological sample 215 immersed in an aqueous liquid medium 210, and the biological sample 215 is being observed by a microscope 250.

[0061] exist Figure 1 In this specification and the appended claims, a vertical direction 80, aligned with the direction of gravity, is defined. Positional and directional terms such as "above," "below," "higher," "upper part," "lower part," "top," "bottom," and "horizontal" are interpreted with reference to the vertical direction 80. It should be noted that, as... Figure 1 The sample container 100 is depicted with its orientation 80° along the vertical direction, and this orientation is generally followed when using the sample container 100 for microscopic observation.

[0062] like Figure 1 and Figure 2 As shown, the sample container 100 includes a container body 110 and a membrane 150.

[0063] The container body 110 includes a bottom 120, sidewalls 130, and a cavity 115. The cavity 115 is a space defined by the sidewalls 130 and the bottom 120. Notably, the cavity 115 is an internal cavity located on the bottom 120 and defined by the sidewalls 130. The cavity 115 is used to contain liquid media 210 and biological samples 215. An aperture 125 is formed in the bottom 120. Furthermore, the aperture 125 extends through the bottom 120. The aperture 125 can have any size that a person skilled in the art would consider suitable. The edge 126 of the aperture 125 may be close to the edge 121 of the bottom 120. Although... Figure 1 The following description uses a hole 125 on the bottom 120 as an example, but this disclosure is not limited to having only one hole on the bottom 120; the bottom 120 may also have multiple holes (for example, in the porous sample container 100a described later).

[0064] A thin film 150 is attached to the bottom 120 to completely cover the aperture 125, thereby forming a cavity-facing surface 155 on the thin film 150 exposed to the cavity 115. Notably, the boundary of the cavity-facing surface 155 on the thin film 150 coincides with the edge 126 of the aperture 125, since the cavity-facing surface 155 is exposed to the cavity 115. It should also be noted that the thin film 150 is attached to the bottom 120 to completely cover the aperture 125, thereby sealing the bottom 120 with the thin film 150 to prevent the liquid medium 210 from leaking through the aperture 125 to the outside of the sample container 100. The cavity-facing surface 155 allows the liquid medium 210 to directly contact the thin film 150, such that the light beam 225 forming the image of the biological sample 215 is transmitted from the liquid medium 210 through the thin film 150 to the microscope 250 without frequently contacting the bottom 120. (To avoid confusion, the image of biological sample 215 is also referred to as 225 below.) Advantageously, film 150 is an FEP film made of FEP, and the cavity-facing surface 155 is an activated surface that enhances the adhesion of the cavity-facing surface 155 to biological sample 215. Due to activation, the adhesion of the cavity-facing surface 155 to biological sample 215 is greater than that of the initial pristine surface of film 150 to biological sample 215. Through these two advantageous features of sample container 100, biological sample 215 is able to adhere to cavity-facing surface 155 compared to conventional glass-bottom containers, and the image resolution of image 225 acquired by microscope 250 is improved (due to the close refractive index between FEP and water). It is important to attach the biological sample 215 to the cavity-facing surface 155 because: (1) it allows the biological sample 215 to grow to a size suitable for microscopic observation by means of adherent cell culture; and (2) since the biological sample 215 is fixed to the cavity-facing surface 155 during microscopic observation, it makes microscopic observation operations more convenient.

[0065] In some implementations, the cavity-facing surface 155 is activated such that the adhesion of the cavity-facing surface 155 to the biological sample 215 is at least comparable to that of the glass surface. Figure 5 (Details will be provided later) An embodiment is provided in which the activated FEP film surface has a higher adhesion to biological cells than a glass surface.

[0066] Further details of sample container 100 are described below.

[0067] The container body 110 can be made of any material, which can be a composite or non-composite material. As a conventional material choice for forming a sample holding container, the container body 110 can be made of glass. Alternatively, the container body 110 can be made of fused silica (also known as fused quartz). The sample container 100 can also be made of other materials that those skilled in the art deem suitable, such as 3D printing materials. Examples of 3D printing materials include PLA and ABS. In addition to transparent materials, non-transparent materials such as plastics and metals can also be selected to form the container body 110.

[0068] In most practical applications, the thickness of film 150 can be from 1 nm to 5 mm. As a trade-off between achieving high transparency of film 150 (which supports lower film thickness values) and providing sufficient mechanical strength for film 150 (which supports higher film thickness values), the inventors have determined that the thickness of FEP film 150 is preferably from 0.025 mm to 0.05 mm (inclusive).

[0069] Thin film 150 also includes an outward-facing surface 151 opposite to the cavity-facing surface 155. In an inverted microscope, where microscope 250 is an inverted microscope and the biological sample 251 is observed from below sample container 100, a refractive matching solution / material 280 matching the refractive index of thin film 150 can be filled between sample container 100 and microscope 250 to avoid additional distortion to image 225 when image 225 of biological sample 215 exits thin film 150 through outward-facing surface 151. Filling refractive matching solution / material 280 between sample container 100 and microscope 250 means filling the space between outward-facing surface 151 of thin film 150 and objective tip 251 of microscope 250. Note that refractive matching solution / material 280 is in close contact with outward-facing surface 151 and objective tip 251. Since FEP and water have similar refractive indices, water can be conveniently used as refractive matching solution / material 280. The membrane 150 also serves to protect the biological sample 215 from the influence of the refractive matching solution / material 280, which may be water contaminated with unknown microorganisms or unknown proteins.

[0070] As described above, plasma treatment can be used to activate the cavity-facing surface 155 to enhance its adhesion to the biological sample 215. Figure 5 This demonstrates the increase in adhesion. Figure 5 The culture results of CT26 cells on (a) glass, (b) plasma-treated FEP films, and (c) untreated FEP films are depicted. Image 510 shows the cell density of CT26 cells on the glass, which is approximately 390 cells / mm². 2Image 520 shows the cell density of CT26 cells on a plasma-treated FEP film, at approximately 410 cells / mm². 2 Image 530 shows the cell density of CT26 cells on a pure, untreated FEP film, at approximately 20 cells / mm². 2 . Figure 5 The results showed that untreated FEP films could not fix biological samples, while plasma-treated FEP films could adhere to biological samples.

[0071] In some implementations, the cavity surface 155 is activated by plasma treatment. The plasma treatment is performed in a gas-filled environment.

[0072] The workflow for producing an FEP film 150 with an activated cavity-facing surface 155 is as follows: The FEP film 150 is subjected to plasma treatment at room temperature. Ionized gas is generated by low-voltage discharge. Under the influence of an electric field, free electrons in the gas gain energy from the electric field and become high-energy electrons. These high-energy electrons collide with atoms and molecules in the gas. Assuming that the energy of an electron is greater than the excitation energy of a molecule or atom, the collisions produce excited-state molecules, excited-state atoms, free radicals, ions, and rays of varying energies. The active ions in the low-temperature plasma generally have energies close to or exceeding the bond energies of carbon-carbon or other carbon-containing bonds, allowing these active ions to interact with gas or solid surfaces. Argon (Ar) plasma can significantly increase the contact angle and surface energy of the FEP film surface through surface physical changes caused by surface crosslinking and etching, thereby improving the surface adhesion of the FEP film 150. The operating conditions for Ar plasma treatment are as follows: the sample container 100 is placed in a chamber with a vacuum degree of 0 to 1 kPa; Ar gas with a purity of 90% to 100% is used at a flow rate of 0.1 to 1 L / min; the plasma power is 5 to 300 W; and the treatment duration is 0.5 to 10 minutes.

[0073] Although the above-described workflow uses Ar gas in plasma processing as an example, the gas used in plasma processing can be a rare gas selected from Ar, helium, neon, krypton, xenon, and radon. Other gases, not limited to rare gases, can also be used in plasma processing.

[0074] Although the workflow uses Ar gas with a purity of 90% to 100% as an example, more generally, the gases used in plasma processing are only required to have a purity level of less than or equal to 100%.

[0075] Although a flow rate of 0.1 to 1 L / min is used as an example in the workflow, more generally, the flow rate of the gas used in plasma processing can be between 0.001 L / min and 10 L / min (inclusive).

[0076] Although the workflow is illustrated using a pressure of 0–1 kPa as an example, more generally, the pressure of the gas used in plasma processing may be as high as 10 MPa.

[0077] Although a plasma power of 5–300 W is used as an example in the workflow, more generally, a power between 1 mW and 5000 W (inclusive) can be used to excite the plasma process.

[0078] While the workflow is illustrated with an example of a plasma treatment time of 0.5 to 10 minutes, more generally, plasma treatment can be performed for a duration of up to 24 hours.

[0079] It is worth noting that the plasma treatment workflow described above alters both the cavity-facing surface 155 and the outward-facing surface 151, activating both to enhance adhesion to the biological sample 215. However, the outward-facing surface 151 is not required to adhere to the biological sample 215. Furthermore, although plasma treatment aims to alter the surface at the molecular level, improper process control may lead to increased surface roughness. Increased surface roughness of the outward-facing surface 151 may adversely affect the resolution of the image 225 of the biological sample 215 during microscopy 250 observation, resulting in decreased resolution. To avoid this problem, the outward-facing surface 151 can be covered with a protective layer during plasma treatment. In some embodiments, the outward-facing surface 151 is the untreated, pristine surface of the film 150. Therefore, the cavity-facing surface 155 exhibits stronger adhesion to the biological sample 215 than the outward-facing surface 151.

[0080] Figure 3 A porous sample container 100a is depicted, which is one embodiment of the disclosed sample container and a variation of sample container 100. The porous sample container 100a is identical to sample container 100 except that it has multiple independent pores 125-1:10 on its bottom 120 instead of a single pore. The multiple independent pores 125-1:10 expose the cavity-facing surface 155, thus forming a plurality of pores. The presence of the multiple independent pores 125-1:10 allows the porous sample container 100a to additionally function as a porous cell culture plate for culturing multiple isolated biological samples before observation through a microscope 250. The multiple isolated biological samples collectively form a biological sample 251.

[0081] exist Figure 2 and Figure 3 In the disclosed sample container, sample container 100 and porous sample container 100a have circular and rectangular shapes, respectively, when viewed from the top. This disclosure is not limited to these two shapes in the formation of the disclosed sample containers. Any shape deemed suitable by those skilled in the art may be used for the disclosed sample containers. Furthermore, the disclosed sample containers may have any size deemed suitable by those skilled in the art.

[0082] Typically, the container body 110 of the sample container 100 or the porous sample container 100a is a rigid body of arbitrary shape. In some embodiments, the container body 110 has a circular or rectangular shape when viewed from above.

[0083] The following two illustrative examples illustrate the manufacture of sample container 100 and its application in cell observation.

[0084] Example 1

[0085] Step 1 - Fixing the FEP film 150. A 0.05 mm thick FEP film 150 is attached to the bottom 120 of a culture dish (as the container body 110), the bottom 120 of which has a hole 125. The hole 125 of the culture dish is sealed by the FEP film 150. The culture dish is used to fix and culture biological samples 215.

[0086] Step 2 - Treat the inner and outer surfaces of the FEP film 150 and the culture dish with Ar plasma. Place the culture dish and FEP film 150 into a vacuum chamber, and introduce 99% pure Ar gas into the vacuum chamber at a flow rate of 0.2 L / min; set the power of the plasma generator to 175 W, and the treatment duration to 2 minutes.

[0087] Step 3 - Fixation and Culturing of Biological Samples 215. The culture dish is sterilized with a UV light source for 15 minutes. After cell culture in sample container 100, the cells are firmly adhered to and fixed onto the FEP film 150. After 24 hours of culturing, sample container 100 can be easily transferred and fixed onto microscope 250. The culture dish can be immersed in water or solution for observation using a high-resolution light-sheet microscope system.

[0088] Step 4 - Observe biological sample 215 using a light-sheet microscope. Immerse the bottom 120 of the culture dish with the FEP film 150 in the solution, and observe the biological sample 215 fixed on the FEP film 150 using a light-sheet microscope.

[0089] Example 2

[0090] Step 1 - Fixing the FEP film 150. A 0.05 mm thick FEP film 150 is installed on the bottom 120 of the sample container 100. The FEP film 150 is fixed between the circular support and the sample container 100 by friction. The sample container 100 is used to fix and culture biological samples 215.

[0091] Step 2 - Treat the inner and outer surfaces of the FEP film 150 and the sample container 100 with Ar plasma. Place the sample container 100 in a vacuum chamber and introduce 99% pure Ar gas into the vacuum chamber at a flow rate of 0.1 L / min. Set the plasma generator power to 150 W and the treatment duration to 2 minutes.

[0092] Step 3 - Fixation and Culturing of Biological Sample 215. The sample container 100 is sealed in a sealed bag and transferred to the biological laboratory. The sample container 100 is allowed to stand for 2 hours. The sample container 100 is small enough to fit into a 12-well plate, and is sterilized with a UV light source for 15 minutes. After cell culture in the plate, the cells adhere firmly to and are fixed onto the FEP film 150. After 24 hours of culturing, the sample container 100 can be easily transferred and fixed onto the microscope 250. The sample container 100 can be immersed in water or solution for high-resolution observation and to protect the biological sample 215 from unknown solutions.

[0093] Control group experiment

[0094] In the control group, ordinary glass slides with a thickness of 0.1 mm and a diameter of 5 mm were used for cell culture fixation and processing. After performing the same sterilization method as in Example 1, the slides were placed directly into 12-well cell culture plates. After culturing for 24 hours, the slides were immersed in water. The differences between the glass slides and sample containers 100 in terms of cell culture status and biological sample image resolution were compared.

[0095] The cell density on the sample container 100 is similar to that on the glass slide, but the image resolution of the cells in the sample container 100 is higher than that of the cells on the glass slide. Because the FEP film 150 is very thin and its diffraction index is close to that of water, the image 225 acquired by the microscope 250 is not distorted and has a higher resolution than that of the glass slide.

[0096] It should be noted that the advantages of sample container 100 are summarized as follows.

[0097] 1. The FEP film 150 can be easily and stably fixed directly to the container body 110 without damage.

[0098] 2. FEP has a diffraction index similar to that of water, thereby improving the image resolution of biological sample 215 in microscopic observation.

[0099] 3. Because the sample container 100 is small in size and easy to use, it can be directly placed into a 6-well or 12-well plate for cell culture.

[0100] 4. The manufacturing process of the above sample container 100 is simple and has good operability.

[0101] 5. The sample container 100 can be directly fixed under the microscope 250, and the operation is convenient.

[0102] 6. The sample container 100 has good mechanical properties and corrosion resistance.

[0103] 7. The disclosed workflow for manufacturing the sample container 100 is not complicated and can keep the manufacturing cost of the sample container 100 low.

[0104] A second aspect of this disclosure is to provide an optical microscope system using the disclosed sample container.

[0105] refer to Figure 2 An exemplary optical microscope system 200 includes a microscope 250 (which is an optical microscope (also labeled 250)) and a sample container 100 for holding a biological sample 215 for observation by the optical microscope 250. Since a porous sample container 100a is also an embodiment of the disclosed sample container, the optical microscope system 200 can alternatively be configured by including both the optical microscope 250 and the porous sample container 100a. In the following description, the optical microscope system 200 having the sample container 100 is used as a representative example of an optical microscope system.

[0106] The optical microscope system 200 can be an upright microscope system, an inverted microscope system, or a tilting microscope system. As an example, Figure 2 The optical microscope system 200 shown is an inverted microscope system.

[0107] Fluorescence microscopy is particularly important for cell biology. In cell studies, fluorescence microscopy is often used to observe subcellular structures to reveal the precise location, development, and function of tissues and cells. In some embodiments, the optical microscope 250 is a fluorescence microscope.

[0108] Figure 4 An optical microscope system 200 is described as a fluorescence microscope system 400 including a fluorescence microscope 410. Although Figure 4The fluorescence microscope 410 described herein is illustrated using a tilting microscope as an example, but the following description also applies to upright and inverted microscopes. The fluorescence microscope 410 includes an excitation optics system 412 and a detection optics system 414. The excitation optics system 412 is configured to generate an excitation beam 415 and irradiate the biological sample 215 with the generated excitation beam 415, thereby exciting the biological sample 215 to fluoresce, resulting in a fluorescence image 418 of the biological sample 215. The detection optics system 414 is configured to capture and amplify the fluorescence image 418, thereby forming a microscope image of the biological sample 215. To demonstrate the practical advantage of imaging the biological sample 215 to achieve enhanced image resolution, the biological sample 215 is immersed in an aqueous liquid medium 210, and a refractive matching solution / material 280 (typically water) fills the space between the thin film 150 and the detection optics system 414.

[0109] The excitation beam 415 selected for the fluorescence microscopy system 400 can be a visible beam, or more commonly a UV beam. However, this disclosure is not limited to using visible light or UV as the excitation beam 415. In this disclosure, the excitation beam 415 can have any wavelength that a person skilled in the art deems suitable.

[0110] Although Figure 4 The example given is that the excitation beam 415 passes through the thin film 150 to irradiate the biological sample 215. However, the excitation optical system 412 can also be positioned such that the excitation beam 415 directly irradiates the biological sample 215 from the top of the opening of the container body 110, or the excitation beam 415 passes through the side wall 130 from the outside of the sample container 100 to the biological sample 215.

[0111] The fluorescence microscope 410 can be a confocal microscope.

[0112] The fluorescence microscope 410 can also be a light-sheet microscope. Therefore, the resulting excitation beam 415 is an excitation light sheet. Generally, the thickness of the excitation light sheet (also labeled 415) can be from 1 nm to 1 mm. If UV light is used as the excitation beam 415, then the excitation light sheet 415 is a UV light sheet. Similarly, if visible light is used as the excitation beam 415, then the excitation light sheet 415 is a visible light sheet.

[0113] One advantage of light-sheet microscopy is that when a biological sample 215 is irradiated by an excitation light sheet 415, the position of the excitation light sheet 415, generated by the excitation optics system 412, can be controlled to perform 3D characterization of the biological sample 215. Specifically, different layers of the biological sample 215 are sequentially irradiated using an excitation light sheet 415 with a very small thickness (e.g., about 1 nm). Therefore, each irradiation excites one layer of the biological sample 215. The excited layer produces fluorescence and forms a fluorescence image 418, which is then captured by the detection optics system 414. By obtaining multiple fluorescence images through imaging different layers, a complete 3D characterization of the biological sample 215 can be obtained. Figure 2 A diagram illustrating the process of generating the 3D characterization is provided. The biological sample 215 is presented as a structure consisting of four layers 215a-215d. The excitation optical system 412 projects light from the light sheet 415 sequentially onto the top layer 215a, the second highest layer 215b, the third highest layer 215c, and the bottom layer 215d, causing the four layers 215a-d to be irradiated and excited sequentially. The detection optical system 414 captures four fluorescence images generated at different times by the four layers 215a-215d.

[0114] exist Figure 4 In this system, the fluorescence microscope system 400 is a tilting microscope system with a light-sheet microscope. One advantage of using a tilting microscope system to observe the biological sample 215 when it is excited by UV light is that it reduces the manufacturing cost of the sample container 100. In upright or inverted microscopes, a horizontal light-sheet that generates UV light from the excitation optics is used to excite the biological sample 215. This horizontal light-sheet needs to pass through the sidewall 130 of the sample container 100. Quartz, capable of transmitting UV light, is needed to form the container body 110. On the other hand, glass, which is opaque to UV light, is not used to form the container body 110. Quartz-based sample containers are more expensive than glass-based sample containers. Under a tilting microscope, a UV light-sheet 415 is tilted into the cavity 115 through an FEP film 150 that transmits both UV and visible light. Therefore, glass can be used to form the container body 110. This reduces the cost of the sample container 100.

[0115] In some embodiments, the excitation optics system 412 and the detection optics system 414 are controllably movable relative to the sample container 100, thereby allowing the four different layers 215a-215d of the biological sample 215 to be sequentially irradiated by the excitation light sheet 415 and subsequently imaged by the detection optics system 414. Thus, the 3D structure of the biological sample 215 can be obtained.

[0116] This disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, embodiments of this disclosure should be considered illustrative rather than restrictive in all respects. The scope of this disclosure is defined by the appended claims rather than the foregoing description, and is therefore intended to cover all changes in the equivalent meaning and scope of the claims.

Claims

1. A sample container for holding a biological sample for microscopic observation, wherein the biological sample is immersed in an aqueous liquid medium during the microscopic observation, the sample container comprising: The container body includes a bottom and a cavity located at the bottom for containing the liquid medium and the biological sample, and the bottom is formed with at least one pore; and A thin film is attached to the bottom to completely cover the at least one aperture, such that a cavity-facing surface is formed on the film, the film including an outward-facing surface opposite to the cavity-facing surface, the cavity-facing surface allowing the liquid medium to directly contact the film, such that a light beam forming an image of the biological sample is transmitted from the liquid medium through the film to the microscope without passing through the bottom, wherein the film is an FEP film made of fluorinated ethylene propylene copolymer (FEP), the cavity-facing surface is an activated surface that enhances the adhesion of the cavity-facing surface to the biological sample, thereby enabling the biological sample to adhere to the cavity-facing surface compared to a conventional glass-bottom container, and improving the image resolution of the image acquired by the microscope due to the close refractive index between FEP and water.

2. The sample container according to claim 1, wherein the container body is made of any material.

3. The sample container according to claim 1, wherein the container body is made of glass, fused silica or any 3D printing material.

4. The sample container of claim 1, wherein the cavity-facing surface is activated such that the adhesion of the cavity-facing surface to the biological sample is at least comparable to that of a glass surface.

5. The sample container of claim 1, wherein the cavity-facing surface is activated by plasma treatment.

6. The sample container of claim 5, wherein the plasma treatment is performed in a gas-filled environment, the gas being a rare gas selected from argon, helium, neon, krypton, xenon, and radon.

7. The sample container of claim 5, wherein the plasma treatment is performed in a gas-filled environment, the purity level of the gas being less than or equal to 100%.

8. The sample container of claim 5, wherein the plasma treatment is performed in a gas-filled environment, wherein the flow rate of the gas during the plasma treatment is from 0.001 L / min to 10 L / min.

9. The sample container of claim 5, wherein the plasma treatment is performed in a gas-filled environment, the pressure of which during the plasma treatment is at most 10 MPa.

10. The sample container of claim 5, wherein the plasma treatment is performed in a gas-filled environment, during which the gas is excited at a power of 1 mW to 5000 W.

11. The sample container of claim 5, wherein the plasma treatment is performed over a duration of up to 24 hours.

12. The sample container of claim 1, wherein the outward surface is the untreated, pristine surface of the film.

13. The sample container according to claim 1, wherein the container body is a rigid body of arbitrary shape.

14. The sample container of claim 1, wherein the container body has a circular or rectangular shape when viewed from above.

15. The sample container according to any one of claims 1 to 14, wherein the at least one pore is composed of a plurality of independent pores, thereby allowing the sample container to be additionally used as a multi-well cell culture plate for culturing a plurality of mutually isolated biological samples prior to observation by the microscope, the plurality of mutually isolated biological samples collectively forming the biological sample.

16. An optical microscope system, comprising: Optical microscope; and The sample container according to any one of claims 1 to 15 is used to hold biological samples for examination by the optical microscope.

17. The optical microscope system of claim 16, further comprising: A refractive matching solution / material that matches the refractive index of the thin film, wherein the refractive matching solution / material is filled between the sample container and the optical microscope to avoid additional distortion of the image when the image of the biological sample leaves the thin film through the outward surface.

18. The optical microscope system of claim 17, wherein the refractive matching solution / material is selected as water.

19. The optical microscope system according to any one of claims 16 to 18, wherein the optical microscope is a fluorescence microscope, comprising: An excitation optical system is configured to generate an excitation beam and irradiate the biological sample with the excitation beam, thereby exciting the biological sample to produce fluorescence, such that the image of the biological sample is a fluorescence image; and A detection optical system is configured to capture and amplify the fluorescence image, thereby forming a microscopic image of the biological sample.

20. The optical microscope system of claim 19, wherein the excitation beam is an ultraviolet (UV) beam.

21. The optical microscope system of claim 19, wherein the fluorescence microscope is a confocal microscope.

22. The optical microscope system of claim 21, wherein the fluorescence microscope is a light sheet microscope, and wherein the generated excitation beam is an excitation light sheet.

23. The optical microscope system of claim 22, wherein the fluorescence microscope is an upright microscope system or an inverted microscope system.

24. The optical microscope system of claim 22, wherein the fluorescence microscope is further comprising a tilting microscope system.

25. The optical microscope system of claim 22, wherein the excitation optics and the detection optics are controllably movable relative to the sample container, such that the excitation light sheet sequentially illuminates different layers of the biological sample, which are then imaged by the detection optics to obtain the three-dimensional structure of the biological sample.

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