Embryo-like sample loading device of light sheet microscope and living body imaging method

Abstract

The invention provides an X-ray plate microscopic imaging loading device for a living embryo sample and a long-term microscopic imaging method, and belongs to the technical field of X-ray plate microscopic imaging. The loading device mainly comprises an FEP cabin body, a fixing ring, a sealing gasket, a sealing cover, a liquid adding capillary tube, a liquid pumping capillary tube, a gas supply capillary tube and a pressure balance capillary tube. The FEP cabin body is prepared through a forming device. The loading device disclosed by the invention can realize rapid and lossless loading of small samples such as embryo-like samples; the closed design can provide a sterile environment; the liquid adding / pumping capillary tube is matched with the injection pump, and the gas supply / pressure balance capillary tube is matched with the gas controller, so that the replacement of a culture solution and the maintenance of a gas environment required by the living embryo sample culture can be automatically realized, and the normal growth and development of the sample in the X-ray plate microimaging process are ensured. The loading device can be matched with different light-sheet microscopic imaging systems, so that uninterrupted high-resolution microscopic imaging observation of the light-sheet microscope in the living embryo sample development process is realized.

Description

Technical Field

[0001] This invention relates to the field of light-sheet microscopy imaging technology, and more particularly to a loading device for embryonic samples in a light-sheet microscopy imaging system, a molding device required for preparing the loading device, a light-sheet microscopy imaging system including the loading device, and an imaging method using the light-sheet microscopy imaging system. Background Technology

[0002] Light sheet microscopy is a novel microscopic imaging technique. Compared to laser scanning confocal microscopy and conventional wide-field fluorescence microscopy, it offers several advantages, including low phototoxicity, high axial resolution, and a large imaging spatial scale, making it highly suitable for long-term three-dimensional imaging observation of embryonic structures, from single-cell to multicellular structures. However, sample preparation and loading for light sheet microscopy differ significantly from conventional microscopy systems, and the loading device and method directly determine the quality of the obtained microscopic image information.

[0003] Prior art related to or similar to this invention:

[0004] In existing light-sheet microscopy imaging techniques, the primary loading method for micron-sized samples such as organoids and embryos is the embedding method. This involves embedding the sample in 1-4% agarose solution and suspending it in a loading chamber to achieve light-sheet microscopic imaging of these samples. Recent reports have described light-sheet microscopy imaging systems specifically designed for these tiny samples: an open multi-sample loading device and a dual-view light-sheet microscopy imaging system. In this system, embryooids, organoids, and other samples are directly loaded into a top-open FEP (Fluorinated ethylene propylene) chamber. The microscope's optical path is set to tilted bilateral illumination, allowing for bilateral illumination and imaging of the sample at the bottom of the FEP chamber. Simultaneously, the addition of culture medium to the FEP chamber enables long-term in vivo observation and imaging of embryooids and organoids.

[0005] The aforementioned shortcomings of the prior art are as follows:

[0006] The embedding method can stably load samples into a microscopic system, but it has the following drawbacks: 1) This method requires agarose embedding of the sample, and after embedding, it needs to be placed in a capillary tube or attached to a sample holder, so the entire sample pretreatment operation is cumbersome and time-consuming; 2) During the agarose embedding and attachment process, the sample may be subjected to external forces such as compression, which can easily damage the original three-dimensional structure of the sample, especially for embryonic samples with internal cavities, where cavity collapse is very likely to occur during the embedding operation; 3) The processing flow and imaging method of this method determine that it is only suitable for imaging dead embryonic samples and cannot achieve long-term dynamic observation of living embryonic samples.

[0007] The open-type multi-sample loading device and dual-view light-sheet microscopy imaging system can achieve long-term in vivo imaging of tiny samples such as embryo-like organisms and obtain high-quality imaging results, but it has the following shortcomings: 1) This sample loading device is a dedicated device for this model of light-sheet microscope and is only suitable for light-sheet microscopes with tilted bilateral illumination, specifically for tiny samples such as organoids and embryo-like organisms; the original intention of light-sheet microscopy technology was to enable rapid imaging of samples from micrometers to centimeters, therefore most light-sheet microscopes on the market are parallel bilateral or unilateral illumination and cannot be adapted to this sample loading device; 2) When performing in vivo imaging, this method requires... Changing the culture medium requires removing the sample loading device from the imaging system, using a pipette tip to aspirate the waste liquid, and then adding new culture medium. This process is: a. rather cumbersome; b. requires interruption of microscopic imaging observation; c. the disassembly, assembly, and movement of the sample loading device, as well as the aspiration and addition of culture medium during the process, may alter the three-dimensional spatial position and angle of the sample, thus drastically increasing the difficulty and cost of integrating and unifying long-term imaging data (such as tracking embryonic cell differentiation); 3) the design of the sample loading device and the imaging method cannot provide the sterile environment required for embryonic cell culture. Summary of the Invention

[0008] In view of this, in order to overcome the above-mentioned shortcomings of the existing technology, in a first aspect, the present invention provides an embryo-like sample loading device for a light sheet microscopy imaging system. The device, through the FEP chamber obtained by the molding device, can realize the rapid and non-destructive loading of tiny samples such as embryos; the sealed design can provide a sterile environment; the design of liquid addition capillary, liquid extraction capillary, gas supply capillary and pressure balancing capillary can realize the precise control and maintenance of culture medium and gas environment in the sample loading FEP chamber, ensuring long-term culture and uninterrupted microscopic imaging observation of the sample.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a loading device for embryonic samples in a light-sheet microscopy imaging system, comprising:

[0011] FEP chamber is used for loading embryo-like samples and culture media required for embryo-like culture;

[0012] The retaining ring is used to abut against the bottom edge of the FEP hull from bottom to top;

[0013] A sealing gasket is provided on the top of the FEP chamber;

[0014] A sealing cap is used to cover the sealing gasket and to tighten the sealing gasket by threading it into the retaining ring.

[0015] The sealing cap has a perforation for the liquid addition capillary, liquid extraction capillary, gas supply capillary and pressure balancing capillary to pass through and extend into the FEP chamber.

[0016] The fixing rod is used to attach and fix the loading device to the imaging chamber of the light sheet microscopy system.

[0017] Secondly, a molding apparatus for preparing the FEP chamber in the loading device includes:

[0018] The base has a through hole at its bottom for connecting to a vacuum pumping device;

[0019] A perforated plate is disposed on the base;

[0020] The FEP cabin male mold is fixed to the upper surface of the perforated plate;

[0021] The FEP foil is located between the upper fixing frame and the lower fixing frame, and is fixed by the upper fixing frame and the lower fixing frame;

[0022] The lower fixing frame is disposed on the perforated plate, and a sealed cavity is formed between the FEP foil and the base.

[0023] Preferably, the FEP cabin male mold is M-shaped with a flat top.

[0024] Thirdly, the present invention provides a light sheet microscopy imaging system for observing embryonic samples, including the above-mentioned loading device;

[0025] The loading device is adapted and fixed to the imaging chamber of the light sheet microscopy system via a fixing rod.

[0026] Fourthly, the present invention also provides a method for performing live imaging of embryonic-like samples using the above-described light-sheet microscopy imaging system, comprising the following steps:

[0027] Embryo-like samples are loaded into the sample slot at the bottom of the FEP chamber of the loading device along with culture medium. After adding a certain amount of culture medium, the device is assembled and sealed. The loading device containing the embryo-like samples is then adapted and fixed into the imaging chamber of the light sheet microscopy system, allowing for direct light sheet microscopy observation.

[0028] The culture medium is automatically circulated and replaced via a liquid addition capillary, a liquid extraction capillary, and a syringe pump. The gas environment is automatically maintained via a gas supply capillary and a pressure balancing capillary, eliminating the need to interrupt imaging.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention provides a loading device specifically designed for light-sheet microscopy systems, particularly suitable for processing tiny samples such as embryos. The device's sealed, independent chamber design effectively solves the problem of suspending and fixing tiny samples in light-sheet microscopy. The sample loading process is fast, convenient, and non-destructive: simply inject the sample along with the culture medium into the FEP chamber of the loading device using a pipette or tube, and then adapt and fix the loading device to the light-sheet microscopy system to complete the sample loading and imaging observation. A groove is provided at the bottom of the loading device chamber, allowing the sample to fall into it under gravity, facilitating sample positioning and maintaining stable spatial position and tilt angle during imaging observation. The loading device chamber is made of FEP (fluorinated ethylene propylene) film, which has high light transmittance and a refractive index compatible with aqueous systems, ensuring unobstructed observation of the illumination and fluorescence collection light paths. Simultaneously, the entire loading device can be used with the light-sheet imaging chamber to form a fully immersive light path, eliminating interfacial light scattering losses between excitation light and fluorescence, while simultaneously controlling and maintaining the temperature of the culture system within the chamber through heat transfer via the liquid system. The sealed design of this device effectively prevents contamination of the culture system. Simultaneously, the loading device is equipped with an automated culture medium replacement system and a gas environment maintenance system. Through the automatic operation of the liquid addition capillary, liquid extraction capillary, and injection pump, the circulation, renewal, and maintenance of the culture medium within the chamber can be achieved. Through the gas supply capillary, pressure balancing capillary, and gas controller, the gas environment within the chamber can be maintained and adjusted. The culture medium replacement system and the gas environment maintenance system together achieve precise control and maintenance of the embryo-like sample culture system within the FEP chamber, ensuring long-term sample culture and uninterrupted, stable microscopic imaging observation. In summary, the sample loading device provided by this invention demonstrates efficient and non-destructive sample loading advantages in processing small samples such as organoids and embryo-like organisms in light-sheet microscopy imaging systems, and can maintain the system activity of live embryo-like samples for extended periods.

[0031] The light-sheet microscopy imaging system and embryonic-like in vivo imaging method involved in this invention are widely applicable to various conventional light-sheet microscopes. Specifically designed to support 5x, 20x, and 40x objectives, it enables high-resolution three-dimensional imaging of embryonic-like samples. Utilizing this innovative technology, researchers can perform long-term, three-dimensional in vivo observation and imaging of embryonic-like (and other types of multicellular systems such as organoids) samples, from single-cell to multicellular levels. This imaging system not only improves imaging resolution but also greatly expands the ability to monitor in vivo samples over long periods in biomedical research, thus providing a powerful tool for research in cell biology, developmental biology, and other related fields. Attached Figure Description

[0032] Figure 1 A breakdown diagram of the loading device;

[0033] Figure 2 This is the main view of the loading device;

[0034] Figure 3 This is a breakdown diagram of the molding device;

[0035] Figure 4 This is a structural schematic diagram of the FEP cabin male mold;

[0036] Figure 5 This is the front view of the molding device;

[0037] Figure 6 A schematic diagram of the loading device being adapted and fixed in the imaging chamber of the light sheet microscopy system.

[0038] In the diagram, 1. FEP chamber; 2. Fixing ring; 3. Sealing gasket; 4. Sealing cap; 5. Liquid filling capillary; 6. Liquid extraction capillary; 7. Gas supply capillary; 8. Pressure balancing capillary; 9. Fixing rod; 10. Base; 11. Orifice plate; 12. FEP chamber male mold; 13. Lower fixing frame; 14. FEP foil; 15. Upper fixing frame. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] In the description of this invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top / bottom", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0041] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "sleeved / connected," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0042] like Figure 1-2 As shown, the present invention provides a loading device for embryonic samples in a light-sheet microscopy imaging system, comprising:

[0043] FEP chamber 1 is used for loading embryo-like samples and culture media required for embryo-like culture;

[0044] The fixing ring 2 is used to abut against the bottom edge of the FEP cabin 1 from bottom to top;

[0045] Sealing gasket 3 is disposed on the top of the FEP chamber;

[0046] The sealing cap 4 is used to cover the sealing gasket 3 and to press the sealing gasket 3 tightly by threading the fixing ring 2;

[0047] The sealing cap 4 has a perforation for the liquid addition capillary 5, liquid extraction capillary 6, gas supply capillary 7 and pressure balancing capillary 8 to pass through and extend into the FEP chamber 1.

[0048] The fixing rod 9 is used to fit and fix the loading device to the imaging chamber of the light sheet microscopy system.

[0049] When using the loading device of the present invention, the embryo-like sample is transferred to the FEP chamber 1 along with the culture medium or buffer solution using a pipette or pipette, and the sample falls into the groove at the bottom of the FEP chamber 1 by gravity; the FEP chamber 1 loaded with the embryo-like sample is inserted into the fixing ring 2 from the bottom, so that the top edge of the FEP chamber 1 is locked to the top surface of the fixing ring 2, a sealing gasket 3 is placed on the top surface of the FEP chamber 1, and finally the sealing cap 4 is used to close and tighten, thus completing the assembly of the loading device.

[0050] The loading device provided by this invention is simple and quick to assemble, and can be adapted to conventional light-sheet microscopy imaging systems; it can realize rapid and non-destructive loading of samples such as embryos; it can provide a closed sterile culture environment; it can realize automated and low-disturbance culture system control and maintenance; and it can realize long-term light-sheet microscopy imaging observation of live embryos and other samples.

[0051] like Figure 3-5 As shown, the present invention provides a molding apparatus for manufacturing an FEP cabin 1, comprising:

[0052] The base 10 has a specially designed through hole on its bottom to facilitate connection and communication with a vacuum pump. Among many possible designs, the through hole is preferably a threaded hole, which provides a more secure and reliable connection while also facilitating disassembly and maintenance when needed.

[0053] A perforated plate 11, mounted on the base 10, is used to securely fix the FEP cabin male mold 12. During implementation, the perforated plate 5 is connected and fixed to the lower fixing frame 4 through the perforations at its bottom. This design not only ensures the stability of the structure but also facilitates assembly and disassembly, thereby improving the overall efficiency and ease of operation.

[0054] The FEP chamber male mold 12 is fixed to the upper surface of the perforated plate 11 with screws and is used to form the FEP chamber 1. The top protrusion of the FEP chamber male mold 12 is used to form the sample groove at the bottom of the FEP chamber 1. The top protrusion design of the FEP chamber male mold 12 can be adjusted according to the size and shape of the sample, and multiple sample grooves can be set as needed, which is suitable for loading and imaging samples of different sizes. Multiple FEP chamber male molds can be fixed as needed for batch preparation of FEP chambers.

[0055] The FEP foil 14 is positioned between the upper fixing frame 13 and the lower fixing frame 15, and its stable fixation is achieved through these two fixing frames. The lower fixing frame 13 is precisely positioned above the perforated plate 11, ensuring that a tight sealed cavity is formed between the FEP foil 14 and the base 10. The advantages of this design are mainly reflected in the following two aspects:

[0056] First, the FEP foil 14 is securely positioned between the upper fixed frame 13 and the lower fixed frame 15. This design not only enhances the stability of the FEP foil 14 but also effectively prevents its displacement or deformation during operation, thereby ensuring the normal operation and accuracy of the equipment.

[0057] Secondly, the lower fixing frame 13 is precisely positioned above the perforated plate 11. This attention to detail ensures that a tight sealed cavity can be formed between the FEP foil 14 and the base 10. This sealed cavity design provides a strong guarantee for the vacuum adsorption forming of the FEP chamber 1 by the FEP foil 14 and the long-term stable operation of the equipment.

[0058] In this invention, the FEP cabin 1 is preferably manufactured according to the following method: Figure 3-5 All components are secured as shown, ensuring the airtightness of the cavity between the FEP foil 14 and the base 10. The FEP foil 14 near the male mold 12 of the FEP chamber is heated and softened using a heat sealer. Then, under vacuum conditions, the FEP foil 14 is deformed and attached to the surface of the male mold 12. Finally, the vacuum is released and heating is stopped. After the FEP foil cools and sets, it is demolded, and excess material is trimmed to obtain the FEP chamber 1.

[0059] In this invention, the FEP cabin 1 is M-shaped with a flat bottom, making it easy to assemble with the aforementioned loading device.

[0060] like Figure 6 As shown, the present invention provides a light sheet microscopy imaging method for observing embryonic samples, including the loading device described above;

[0061] The loading device, which contains the embryo-like sample, is fixed and adapted to the imaging chamber of the light-sheet microscopy system via a fixing rod 9, allowing the loading device to suspend within the imaging chamber. By adding a refractive index matching solution (water, buffer solution, or culture medium can be used) to the imaging chamber of the light-sheet microscopy system, direct light-sheet microscopic imaging observation of the embryo-like sample can be performed. The fixing rod 9 can be adjusted and adapted to fit different light-sheet microscopy systems.

[0062] Fourthly, the present invention also provides a method for performing live imaging of embryonic-like samples using the above-described light-sheet microscopy imaging system, comprising the following steps:

[0063] Add a refractive index matching solution (water, buffer solution, or culture medium can be used) to the imaging chamber of the light sheet microscopy system, and preheat the refractive index matching solution to 37°C through the imaging chamber of the light sheet microscopy system.

[0064] The loading device containing the embryo-like sample is fixed and adapted to the imaging chamber of the light-sheet microscopy system via a fixing rod 9, allowing the loading device to be suspended in the imaging chamber of the light-sheet microscopy system, and the imaging chamber of the light-sheet microscopy system provides the temperature (37°C) required for the culture of the live embryo-like sample.

[0065] The culture medium required to maintain the viability of embryo-like samples inside the loading device chamber is automatically circulated and updated through the liquid addition capillary 5, the liquid extraction capillary 6, and the injection pump. The required specific CO2 / O2 component gas environment is automatically circulated and regulated through the gas supply capillary 7, the pressure balancing capillary 8 with a 0.22μm hydrophobic filter at the outlet section, and the gas controller for premixing gas, without interrupting imaging.

[0066] The advantages of this imaging method include:

[0067] Direct operation under sterile conditions avoids the risk of sample contamination. Using pipettes or tubes to load samples does not damage their structure or composition, and the operation is simple and quick. The FEP chamber 1 is secured by a retaining ring 2 and a sealing gasket 3, ensuring a tight seal and providing the sterile environment required for embryonic culture. The design of liquid addition capillary 5, liquid extraction capillary 6, gas supply capillary 7, and pressure balancing capillary 8 facilitates precise control and maintenance of the microenvironment required for embryonic culture. The culture medium injection pump and the gas supply for the target components are both automatically timed, improving experimental efficiency and meeting diverse experimental needs.

[0068] The above description is merely a preferred embodiment of the present invention. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention should be covered within the scope of protection of the present invention.

Claims

1. A molding apparatus for preparing an FEP chamber required for loading embryonic samples into a light sheet microscopy imaging system, characterized in that, include: The base has a through hole at its bottom for connecting to a vacuum pumping device; A perforated plate is disposed on the base; The FEP chamber male mold is fixed to the upper surface of the perforated plate and has an FEP chamber; The FEP foil is located between the upper and lower fixing frames and is tightly fixed by the upper and lower fixing frames; The lower fixing frame is disposed on the perforated plate, and a sealed cavity is formed between the FEP foil and the base.

2. The forming apparatus for preparing the FEP chamber required for loading embryonic samples into a light sheet microscopy imaging system according to claim 1, characterized in that, The FEP chamber male mold is M-shaped with a flat top and two recesses for sample placement.

3. A loading device for embryonic samples in a light-sheet microscopy imaging system, characterized in that, include: A retaining ring, fitted around the outside of the FEP cabin body, is used to secure the top of the FEP cabin. A sealing gasket is provided on the top surface of the FEP chamber; A sealing cap is used to cover the retaining ring and press the sealing gasket tightly; The sealing cap has a perforation for the liquid addition capillary, liquid extraction capillary, gas supply capillary, and pressure balancing capillary to pass through and extend into the FEP chamber.

4. A light-sheet microscopy imaging system for observing embryonic-like samples, characterized in that, Includes the loading device as described in claim 3; The loading device is adapted and fixed to the imaging chamber of the light sheet microscopy system via a fixing rod; The loading device allows for high-resolution three-dimensional imaging of intact embryonic samples using a 20x objective lens with a working distance as low as 2.5mm.

5. A method for performing live imaging of embryonic-like samples using the light-sheet microscopy imaging system of claim 4, characterized in that, Includes the following steps: Embryo-like samples are loaded into the sample groove at the bottom of the FEP chamber along with culture medium using a pipette or pipette. After adding culture medium, the chamber is assembled and sealed, and then directly observed by light slide microscopy. The culture medium is automatically circulated and replaced via a liquid addition capillary, a liquid extraction capillary, and an injection pump. The gas environment is automatically maintained via a gas supply capillary, a pressure balancing capillary, and a gas controller, without interrupting imaging.

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