A monitoring system and method for a bioreactor

Abstract

The present application relates to the technical field of cell culture monitoring, in particular to a monitoring system and method for a bioreactor. The system comprises a control module and a mounting seat, one side of the mounting seat is provided with a mounting groove, the bottom of the mounting groove is provided with a monitoring window, and the top of the mounting groove is provided with an illumination module for illuminating a near-wall area target; the mounting seat is provided with a mounting cavity in communication with the monitoring window, and the mounting cavity is provided with an optical path refraction assembly along an optical path, an imaging objective, and an imaging module in sequence; the optical path refraction assembly is used for turning a vertical incident light beam to propagate in a horizontal plane; the imaging objective is used for high-resolution magnification imaging of the near-wall area target and generating a corresponding optical image; and the imaging module is used for receiving the optical image and outputting a digital image signal to the control module. The present application can realize real-time, stable, repeatable and traceable non-invasive microscopic monitoring of cell adhesion, aggregate formation and biomass dynamic trend in the near-wall area of the bag without puncturing, modifying or replacing the existing standard disposable cell culture bag.

Description

Technical Field

[0001] This invention relates to the field of cell culture monitoring technology, and more specifically to a monitoring system and method for bioreactors. Background Technology

[0002] Bioreactors, as key equipment supporting core biological processes such as cell culture, microbial fermentation, and enzyme catalysis, are widely used in fields such as biomedicine, food industry, environmental engineering, and bioenergy. Their core function is to provide a precisely controllable physicochemical environment for biological systems, including key parameters such as temperature, pH, dissolved oxygen (DO), mixing methods, nutrient supply, and metabolite removal. In recent years, with the rapid development of Single-Use Technology (SUT), wave-type (also known as rocking) single-use bioreactors have become an important platform for mammalian cell culture—especially shear-sensitive CHO (Chinese hamster ovary) cells or stem cell systems—due to their elimination of the need for cleaning validation, reduced risk of cross-contamination, flexible deployment, and applicability to various scales from R&D to pilot-scale testing. Wave-type bioreactors use an external shaker to drive the flexible, disposable cell culture bag to tilt periodically, creating an internal wave flow to achieve mixing and mass transfer, avoiding the high shear stress associated with traditional agitators. In the context of modern biopharmaceuticals emphasizing process analysis technology (PAT) and the concept of quality by design (QbD), the full-process, traceable, and quantitative monitoring of critical process parameters (CPPs) and critical quality attributes (CQAs) has become a fundamental requirement for regulatory compliance. Especially for adherent cells, microcarrier cultures, or systems prone to aggregation, the dynamic changes in cell adhesion, aggregate formation, and biomass within a thin layer of tens to hundreds of micrometers near the cell wall directly affect product expression efficiency and process robustness, urgently requiring high spatiotemporal resolution visualization methods for capture. Wave-type bioreactor cell culture is typically based on large-volume disposable cell culture bags, which generally integrate sensors for conventional physicochemical parameters such as temperature, pH, DO, and CO2, enabling closed-loop control of the culture environment. However, existing systems generally lack the ability to perceive real-time changes in cell morphology, spatial distribution, and biomass dynamics within the bag, particularly struggling to visualize and monitor key microscopic events such as cell adhesion and aggregate formation in the near-wall region. To fill this gap, researchers have attempted to introduce microscopic imaging technology, currently categorized into invasive and non-invasive approaches. Invasive microsurgical monitoring typically achieves internal imaging by puncturing or embedding miniature optical windows, fiber optic probes, or endoscopes into the reaction bag wall. However, such methods have significant drawbacks: (1) any puncture or rigid embedding structure will compromise the integrity and seal of the disposable system, significantly increasing the risk of contamination; (2) the probe surface is highly susceptible to protein adsorption, cell attachment, or microbubble interference, leading to rapid degradation of imaging quality, signal drift, or sluggish response; (3) under the unique periodic oscillation conditions of wave-type bioreactors, the flexible bag cannot withstand local stress concentration, and forcibly integrating rigid components can easily cause bag membrane fatigue, leakage, or even mechanical failure.Furthermore, invasive devices typically require reuse and sterilization validation procedures, violating the core principle of "use and discard" for disposable technologies and failing to meet the compliance and ease-of-use requirements of Good Manufacturing Practices (GMP). To overcome these limitations, non-invasive microsurgical monitoring technologies have gradually become a research hotspot. For example, patent application CN114729295A provides a cell culture container and monitoring system for non-invasive cell culture monitoring, comprising a dedicated closed cell culture chamber and an external monitoring module, which includes a fusion monitor. The cell culture chamber has an optical window made of polymer material embedded in its wall, allowing the externally mounted fusion monitor to measure cell states (such as density, morphology, or distribution) inside the cell culture chamber without physical contact with the cell culture or culture medium. This approach avoids puncture procedures, effectively maintaining the aseptic integrity of the system, and conceptually represents a significant step towards non-contact, sealed, and compatible monitoring.

[0003] However, the core premise of the aforementioned existing technology is the need to use specially designed, customized cell culture containers with integrated optical windows. This means that the technology cannot be directly adapted to the standard disposable cell culture bags and other components used in wave-type bioreactors widely adopted in the industry. Therefore, although the technology has made progress in aseptic assurance, it has significant limitations in platform versatility—users who want to deploy this monitoring system must replace the entire reaction container, which not only significantly increases equipment and validation costs but also involves complex process transfers and regulatory filings, seriously hindering the large-scale application of this technology on existing disposable biomanufacturing platforms. Summary of the Invention

[0004] The purpose of this invention is to provide a monitoring system and method for bioreactors, which partially solves or alleviates the above-mentioned deficiencies in the prior art. It enables real-time, stable, repeatable and traceable non-invasive microscopic monitoring of cell adhesion, aggregate formation and biomass dynamics in the near-wall region of the cell culture bag without puncture, modification or replacement of existing standard disposable cell culture bags.

[0005] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution:

[0006] A first aspect of the present invention is to provide a monitoring system for a bioreactor for monitoring cell adhesion, aggregate formation, and biomass dynamics in the near-wall region of a cell culture bag; the cell culture bag includes a bag body and sealing strips connected to opposite sides of the bag body, the bag body being used to contain cell culture medium and perform cell culture, and the sealing strips being used to fix the bioreactor.

[0007] The monitoring system includes a control module and a mounting base. One side of the mounting base has a mounting groove for accommodating the side wall of the bag body. A monitoring window is provided at the bottom of the mounting groove, and an illumination module is provided at the top of the mounting groove. The illumination module is electrically connected to the control module and is used to illuminate the target in the near-wall area corresponding to the monitoring window.

[0008] The mounting base has an internal mounting cavity, and the monitoring window communicates with the mounting cavity. Within the mounting cavity, along the optical path, are arranged sequentially: an optical path refraction assembly, including at least one optical path refraction element, for refraction of the vertically incident light beam passing through the monitoring window into a horizontal plane; an imaging objective lens, located on the outgoing optical path of the optical path refraction assembly, for high-resolution magnification imaging of the near-wall region target and generating a corresponding optical image; and an imaging module, electrically connected to the control module, for receiving the optical image generated by the imaging objective lens and outputting a digital image signal to the control module.

[0009] Preferably, as an improvement, the optical path refraction assembly includes a plurality of optical path refraction elements for refraction of the vertically incident light beam passing through the monitoring window into a beam that propagates along the length of the mounting groove in the horizontal plane.

[0010] Preferably, as an improvement, the monitoring system further includes a relay lens group disposed in the optical path within the mounting cavity for relaying the optical image formed by the target in the near-wall region.

[0011] Preferably, as an improvement, the monitoring system further includes an optical filtering device, which includes a polarizer disposed in the light output path of the illumination module, and an analyzer and a wavelength selective filter disposed in the light path within the mounting cavity, located between the imaging objective and the imaging module.

[0012] Preferably, as an improvement, the monitoring system further includes a drive module, and the imaging module is connected to the output end of the drive module; the drive module is electrically connected to the control module and is used to drive the imaging module to move along the optical axis of the imaging objective lens to adjust the imaging distance between the imaging module and the near-wall region target; wherein, the monitoring system further includes a guide component, which is arranged along the optical axis of the imaging objective lens and is used to guide and limit the movement of the imaging module.

[0013] Preferably, as an improvement, the monitoring system further includes a support plate disposed on the side of the mounting base near the cell culture bag; the bottom surface of the support plate is coplanar with the bottom surface of the mounting base, and the top surface slopes downward away from the mounting base and smoothly connects with the bottom of the mounting groove; wherein, transition plates are provided on both sides of the support plate along the length direction of the mounting groove, the top surface of the transition plates slopes downward away from the support plate and smoothly connects with the top surface of the support plate; and / or, the bottom of the mounting base and / or the support plate is provided with an anti-slip pad.

[0014] Preferably, as an improvement, the monitoring system further includes a clamping assembly disposed at the end of the mounting base for clamping and fixing the mounting base onto the sealing strip; wherein the clamping assembly includes a connector and a U-shaped clamp, the U-shaped clamp being connected to the mounting base via the connector, and the opening size of the U-shaped clamp matching the thickness of the sealing strip; wherein an anti-slip strip is provided on the inner wall of the U-shaped clamp; and / or, the U-shaped clamp is slidably connected to the connector in a vertical direction.

[0015] Preferably, as an improvement, a transparent carrier plate is provided inside the monitoring window, and the top surface of the transparent carrier plate is coplanar with the bottom of the mounting groove; wherein, a transparent heating film is provided on the transparent carrier plate; and / or, a flexible attachment pad is provided on the inner wall of the mounting groove; and / or, the lighting module is rotatably connected to the top of the mounting groove; and / or, the lighting module is slidably connected to the top of the mounting groove along the width direction of the mounting groove.

[0016] Preferably, as an improvement, the monitoring system further includes a phase sensor electrically connected to the control module, used to detect the motion phase signal of the bioreactor in real time and output a zero-phase reference point and motion period T; the control module is configured to:

[0017] Within a preset phase scanning window, phase-locked trigger signals are generated sequentially according to a preset phase step, driving the illumination module to emit short pulse light and simultaneously triggering the imaging module to acquire image frames.

[0018] For each phase angle φ, at least two consecutive image frames are acquired to obtain the corresponding pixel matrix; the pixel values ​​of two adjacent frames are subtracted point by point, and the median absolute difference is calculated as the stillness index E(φ) for that phase, i.e.: Where φ represents the phase angle, median represents the median operation, and I t with I t+1 E(φ) represents the pixel value between two adjacent frames; the smaller the value, the smaller the change in the image between adjacent frames, and the more static the image is.

[0019] Based on the above calculation results, the phase angle with the smallest E(φ) is selected as the ideal imaging phase φ. * According to the ideal imaging phase φ * Calculate the trigger delay Δt based on the time relationship between the zero-phase reference point and the reference point.

[0020] During subsequent imaging, the control module synchronously outputs a camera trigger signal to the imaging module and a strobe control signal to the illumination module at intervals of Δt after the zero-phase time t0 of each motion cycle.

[0021] A second aspect of the present invention provides a monitoring method for a bioreactor, implemented based on the aforementioned monitoring system for a bioreactor, comprising the following steps: S1, acquiring the motion phase signal of the bioreactor, determining the zero-phase reference point and the motion period T; S2, within a preset phase scanning window, sequentially generating phase-locked trigger signals according to a preset phase step, driving the illumination module to emit short pulse light, and simultaneously triggering the imaging module to acquire image frames; S3, for each phase angle φ, continuously acquiring at least two image frames to obtain the corresponding pixel matrix; calculating the median absolute difference by subtracting the pixel values ​​of adjacent two image frames point by point, which serves as the stillness index E(φ) under that phase, i.e.: Where φ represents the phase angle, median represents the median operation, and I t with I t+1 E(φ) represents the pixel values ​​of two adjacent frames; the smaller the value of E(φ), the smaller the image change between adjacent frames, and the more static the image tends to be; S4, select the phase angle with the smallest E(φ) as the ideal imaging phase φ. * According to the ideal imaging phase φ * Calculate the trigger delay Δt based on the time relationship between the zero-phase reference point and the zero-phase reference point; S5, in each subsequent motion cycle, after the zero-phase time t0, at an interval of Δt, synchronously output the camera trigger signal to the imaging module and output the strobe control signal to the illumination module.

[0022] Beneficial technical effects of the present invention: The present invention provides a non-invasive near-wall monitoring system and method for wave-type disposable bioreactors, which has significant technical advantages in both structural design and control strategy, and effectively solves the core problem that existing technologies cannot simultaneously achieve sterility, versatility and imaging stability.

[0023] In terms of structural design, this invention fully ensures the aseptic integrity of the system through a completely non-invasive near-wall imaging method. Specifically, the entire monitoring system is integrated inside the mounting base, including an illumination module, optical path refraction components, imaging objective lens, and imaging module. It is positioned externally to the cell culture bag, achieving optical contact only through the monitoring window against the side wall of the bag body. This eliminates the need to puncture the bag, embed a dedicated optical window, or disrupt the original sealing structure of the disposable cell culture bag. Compared to existing technologies that rely on customized containers, this invention can be directly adapted to the standardized wave-shaped disposable cell culture bags widely used in industry. Without altering the original reactor configuration or introducing the risk of contamination, it achieves high-resolution visual monitoring of the near-wall area, significantly improving the system's versatility and ease of industrial deployment.

[0024] Meanwhile, this invention incorporates a light path refraction component within the mounting cavity, redirecting the incident light beam that passes vertically through the monitoring window to propagate horizontally. This allows the imaging objective and imaging module to be arranged horizontally, significantly reducing the system's vertical height requirement. This design effectively prevents mechanical interference between the monitoring device and the shaking table platform, incubator shelves, or surrounding piping under the periodic swaying conditions of a wave-like bioreactor, significantly enhancing the system's adaptability in confined spaces and dynamic environments.

[0025] Furthermore, this invention achieves rapid and detachable fixation of the monitoring system to the bag body through the coordinated operation of the mounting groove, the sealing strip at the edge of the cell culture bag, and the U-shaped clip. This structure not only conforms to the "use and discard" operation concept of disposable technology, but also supports the focusing adjustment of the imaging module along the optical axis through the drive module and guide components, effectively compensating for fluctuations in object distance caused by differences in bag film thickness, installation tolerances, or changes in bag tension during culture, thereby maintaining imaging clarity over a long period of time.

[0026] Building upon this foundation, the present invention further incorporates a polarizer in the illumination optical path and an analyzer and wavelength-selective filter in the imaging optical path, constructing a composite optical suppression mechanism that combines polarization modulation and spectral filtering. This mechanism significantly reduces the interference of reflected light from the bag surface, scattered light from the culture medium, and stray light from the environment on near-wall imaging, greatly improving the signal-to-noise ratio and contrast of images of cells, aggregates, and microcarriers within the near-wall thin layer, providing high-quality raw data support for subsequent quantitative image analysis.

[0027] In terms of control methods, this invention introduces a phase sensor to detect the periodic motion state of the wave-like bioreactor in real time and output a zero-phase reference point. Based on this, the control module generates phase-locked trigger signals sequentially within a preset phase scanning window, driving the illumination module to emit short pulses of light and simultaneously triggering the imaging module to acquire image frames. This mechanism transforms the continuous high-speed movement of the bioreactor into a quasi-static observation process based on phase-discrete sampling, effectively overcoming the motion blur problem caused by periodic oscillations, and enabling clear capture of microscopic dynamic processes such as cell adhesion, aggregate formation, and evolution in the near-wall region.

[0028] Furthermore, this invention constructs a "staticity index" to quantify the degree of stillness in an image by calculating the median absolute difference of pixel values ​​between adjacent image frames. This index statistically evaluates multiple consecutively acquired images at the same phase, effectively suppressing misjudgments caused by abnormal noise or transient disturbances in a single frame using median calculation. The control module automatically identifies the ideal imaging phase corresponding to the minimum value of the staticity index through full-phase scanning and calculates the precise trigger delay accordingly. This method does not rely on prior assumptions of the motion model, nor does it require calibration of the reactor's mechanical structure, and can accurately lock the optimal imaging window under complex fluid disturbance and nonlinear motion response conditions.

[0029] During subsequent imaging, the control module delays the trigger after the zero-phase moment of each motion cycle, synchronously outputting the camera trigger signal and the stroboscopic control signal, forming a stroboscopic imaging mechanism that is strictly phase-locked with the reactor motion. This mechanism ensures strict spatiotemporal consistency of image data acquired across different culture cycles and batches, providing a reliable data foundation for image-based cell growth curve fitting, aggregate particle size distribution statistics, and biomass dynamic trend analysis.

[0030] Furthermore, this invention employs a coordinated control strategy of short-pulse illumination and phase-locked triggering, performing a single exposure only at the ideal imaging phase within each motion cycle. This significantly reduces the cumulative light radiation dose received by cells, effectively avoiding physiological interference caused by long-term illumination to photosensitive cells (such as CHO cells or stem cells). Simultaneously, since a clear image can be acquired in each cycle, the system can accumulate hundreds to thousands of high-quality temporal images on an hourly scale, fully meeting the high-temporal-resolution quantitative analysis requirements for key processes such as cell adhesion rate, aggregation kinetics, and biomass evolution.

[0031] In summary, this invention, through its non-invasive, universal, and modular structural design, combined with a phase-locking mechanism at the control level and an adaptive imaging strategy based on the static degree index, successfully achieves highly reliable, high-resolution, and fully visualized monitoring of near-wall microscopic processes within a standard disposable wave-shaped bioreactor. This fills the gap in current process analysis technology (PAT) for the dynamic perception of cell spatial distribution and provides solid technical support for the intelligentization, data-driven approach, and quality controllability of biopharmaceutical processes. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0033] Figure 1 This is a schematic diagram showing the installation location of the monitoring system provided in this embodiment of the invention within a bioreactor;

[0034] Figure 2 This is a schematic diagram of the connection between the monitoring system and the cell culture bag provided in an embodiment of the present invention;

[0035] Figure 3 for Figure 2 A schematic diagram of the structure after the U-shaped clip slides upward (cell culture bag not shown);

[0036] Figure 4 for Figure 2 A schematic diagram of the structure after the U-shaped clip slides downwards (cell culture bag not shown).

[0037] Figure 5 This is a schematic diagram of the overall structure of the monitoring system provided in an embodiment of the present invention;

[0038] Figure 6 This is a schematic diagram of the connection between the lighting module and the mounting base provided in an embodiment of the present invention;

[0039] Figure 7 A cross-sectional view along the mounting base of the lighting module provided in an embodiment of the present invention;

[0040] Figure 8 for Figure 7 Schematic diagram of the structure at point A;

[0041] Figure 9A cross-sectional view along the longitudinal direction of the mounting base at the imaging module provided in an embodiment of the present invention;

[0042] Figure 10 for Figure 9 A schematic diagram of the structure of the driving module and the imaging module.

[0043] Summary of reference numerals in the attached drawings: Shaker 100, Cell Culture Bag 200, Bag Body 201, Sealing Tape 202; Mounting Base 1, Mounting Slot 11, Monitoring Window 12, Transparent Carrier Plate 13, Slide 14, Support Plate 15, Transition Plate 16, Mounting Cavity 17; Lighting Module 2, Housing 21, LED Light Source 22, Ball Joint 23, Ball Mouth Seat 231, Ball Head 232, Damping Ring 233, Slider 24; Clamping Assembly 3, Connector 31, Transverse Component 311 Vertical component 312, guide groove 313, U-shaped clamp 32, guide block 321, anti-slip strip 33; optical path refraction assembly 4, optical path refraction element 40, first refraction element 41, second refraction element 42, third refraction element 43; imaging objective lens 5; imaging module 6; relay lens group 7; drive module 8, drive motor 81, drive screw 82, drive block 83, guide assembly 84, linear guide rail 841, guide rail block 842, limit stop block 843. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In this document, suffixes such as "module," "component," or "unit" used to represent elements are only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "component," or "unit" can be used interchangeably. In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present 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, and therefore should not be construed as a limitation of the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "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, 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. "And / or" in this document includes any and all combinations of one or more of the listed related items. "Several" or "a plurality of" in this document means two or more, i.e., it includes two, three, four, five, etc. As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%. In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, a range description should be considered as having specifically disclosed all possible subranges and the individual numeric values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0045] Example 1: This example provides a monitoring system for a bioreactor, such as... Figure 1 As shown, the monitoring system is located within the shaker 100 of the wave-type bioreactor, and is used to monitor cell adhesion, aggregate formation, and biomass dynamics in the near-wall region within the cell culture bag 200. Combined with... Figure 2 As shown, the cell culture bag 200 is a standard disposable cell culture bag 200 that is widely used in the current industry for wave-type bioreactors. It includes a bag body 201 and sealing strips 202 connected to opposite sides of the bag body 201. The bag body 201 is used to contain cell culture medium and carry out cell culture, and the sealing strips 202 are used to fix it to the bioreactor.

[0046] like Figure 1 and Figure 5 As shown, the monitoring system includes a control module and a mounting base 1. One side of the mounting base 1 is attached to the side wall of the shaker 100, and the side of the mounting base 1 away from the side wall of the shaker 100 is provided with a mounting groove 11 for accommodating the side wall of the bag body 201.

[0047] In some embodiments, the mounting groove 11 is a U-shaped groove, and the height of the mounting groove 11 is greater than the thickness of the bag body 201. In this way, the sidewalls of the bag body 201 of cell culture bags of different sizes can be smoothly embedded into the mounting groove 11. The bottom of the bag body 201 fits into the mounting groove 11 while a gap is left at the top. This ensures positioning stability and avoids excessive constraint that could damage the flexible bag body 201 during swaying. At the same time, it reduces mechanical interference applied to the outside and ensures that the flow field inside the bag and the cells move naturally according to the original culture mode.

[0048] In some embodiments, a flexible attachment pad (not shown in the figure) is provided on the inner wall of the mounting groove 11. The flexible attachment pad can be made of soft polymer materials with high elasticity, good biocompatibility, smooth surface and chemical inertness, such as medical-grade silicone, thermoplastic polyurethane (TPU), ethylene-vinyl acetate copolymer (EVA) or polydimethylsiloxane (PDMS).

[0049] A flexible attaching pad is added to the inner wall of the mounting groove 11 to prevent the hard contact surface from scratching the bag film, reduce the risk of micro-leakage, and ensure a closed and sterile environment for the disposable system. During the periodic shaking of the shaker 100, the flexible attaching pad can absorb the impact and friction between the bag body 201 and the mounting groove 11, preventing the bag body 201 from wearing, perforating, or failing due to local stress concentration. Furthermore, the flexible attaching pad can fill the tiny gaps between the bag wall and the groove wall through elastic deformation, improving the positioning stability of the bag body 201.

[0050] like Figure 5As shown, a monitoring window 12 is provided at the bottom of the mounting slot 11, and an illumination module 2 is provided at the top of the mounting slot 11. The illumination module 2 is electrically connected to the control module and is used to illuminate the target near the wall area corresponding to the detection window, so as to support optical monitoring of cell adhesion, aggregate formation and biomass dynamics.

[0051] In some implementations, such as Figure 2 , Figure 5 and Figure 7 As shown, a transparent carrier plate 13 is sealed and embedded within the monitoring window 12, and the top surface of the transparent carrier plate 13 is coplanar with the bottom of the mounting groove 11. This ensures that the sidewall of the bag body 201 can fit flat against the monitoring field of view after being inserted into the mounting groove 11, avoiding imaging distortion caused by local depressions or warping. The transparent carrier plate 13 is made of a material with high light transmittance and good biocompatibility. Optional materials include, but are not limited to, borosilicate glass, polymethyl methacrylate (PMMA), polycarbonate (PC), or polydimethylsiloxane (PDMS). Among these, glass is suitable for scenarios with extremely high optical stability requirements; PMMA and PC offer a balance of lightweight and impact resistance, making them suitable for general industrial applications; PDMS, due to its flexible properties, can further buffer the contact stress between the bag body 201 and the transparent carrier plate 13, making it suitable for fragile cellular systems sensitive to mechanical disturbances.

[0052] By incorporating the transparent carrier plate 13, the cell culture bag 200 is provided with localized rigid support, preventing excessive deformation during wave-like swaying that could affect the near-wall flow field or damage the membrane material. Furthermore, it forms a stable optical path, effectively coupling the light beam emitted by the top illumination module 2 and reducing scattering and refraction interference caused by membrane wrinkles, microbubbles, or uneven thickness, thereby significantly improving the imaging signal-to-noise ratio and monitoring repeatability. In addition, the transparent carrier plate 13 is sealed within the monitoring window 12, helping to maintain the overall airtightness of the disposable system, preventing external contamination, and facilitating cleaning or replacement, meeting the GMP requirements for equipment validation and ease of operation.

[0053] In some embodiments, a transparent heating film (not shown) is disposed on the transparent carrier plate 13. Specifically, the transparent heating film is disposed on the side of the transparent carrier plate 13 near the cell culture bag 200, and the top of the transparent heating film is coplanar with the bottom of the mounting groove 11. The transparent heating film is electrically connected to the control module and is configured to controllably raise the temperature of a local area of ​​the transparent carrier plate 13 and the cell culture bag 200 attached thereto during monitoring, in order to compensate for the temperature gradient of the bag wall caused by environmental heat dissipation or thermal resistance of optical components, and maintain the uniformity and stability of the culture medium temperature in the near-wall area.

[0054] The transparent heating film can be made of any of the following high-transmittance conductive materials: (1) Indium tin oxide (ITO) film, deposited on PET or glass substrate, with excellent visible light transmittance (≥85%) and uniform heating characteristics; (2) Fluorine-doped tin oxide (FTO) coating, which is resistant to high temperature and has good chemical stability, and is suitable for scenarios that require repeated cleaning or high-temperature disinfection; (3) Metal nanowire mesh (such as silver nanowire) composite film, which has high conductivity, flexibility and light transmittance, and can adapt to slight deformation without failure; (4) Graphene or carbon nanotube transparent conductive film, which has the advantages of low thermal inertia, fast response and biocompatibility.

[0055] By integrating a transparent heating film, precise local temperature control of the monitoring field of view can be achieved without obstructing the optical path, avoiding abnormal cell adhesion, protein precipitation, or microenvironment disturbance caused by bag wall cooling. Furthermore, this design effectively suppresses condensation on the surface of the transparent carrier plate 13, ensuring imaging clarity and sensor signal stability. In addition, the heating function is dynamically adjusted by the control module based on preset process parameters or real-time temperature feedback, ensuring that the entire culture process meets the GMP requirements for temperature consistency in bioreactors, while maintaining the sterile, sealed structure of the disposable cell culture bag 200.

[0056] Specifically, such as Figure 6 and Figure 8 As shown, the illumination module 2 includes a housing 21 and a high-brightness LED light source 22 disposed within the housing 21. The LED light source 22 is preferably a white LED or a monochromatic LED of a specific wavelength (such as 470nm blue light or 525nm green light) to match the imaging requirements of different cell types or fluorescent labels. Optionally, the illumination module 2 also includes a collimating lens group (not shown) for shaping the divergent beam emitted by the LED light source 22 into a parallel or micro-focused beam to improve illumination uniformity and reduce stray light.

[0057] In some implementations, such as Figure 6 As shown, the lighting module 2 is rotatably connected to the top of the mounting slot 11. For example, the lighting module 2 is connected to the top of the mounting slot 11 via a ball joint 23. Specifically, in conjunction with... Figure 8 As shown, the ball joint 23 includes a ball socket 231 fixedly connected to the top of the mounting groove 11 and a ball head 232 fixedly connected to the top of the housing 21 of the lighting module 2. The ball head 232 is embedded in the ball socket 231 to form a universal joint, allowing the lighting module 2 to achieve multi-degree-of-freedom adjustment in both horizontal and vertical directions. Optionally, the lighting module 2 can automatically lock after rotating to a set angle. Specifically, in conjunction with... Figure 8 As shown, the inner wall of the ball socket 231 is provided with a damping ring 233 made of elastic silicone or polyurethane. When the lighting module 2 rotates to the required angle, the damping ring 233 achieves self-locking through friction, and the lighting optical axis can be kept stable without additional fasteners.

[0058] This structural design allows the illumination module 2 to flexibly adjust the incident illumination direction (such as normal incidence, oblique incidence, or grazing incidence) based on the actual adhesion position of the cell culture bag 200 in the mounting slot 11, the bag thickness, or the depth of the target monitoring area. This optimizes the uniformity of illumination in the near-wall area, reduces surface reflection interference, and enhances the imaging contrast of cell aggregates or adherent layers. For example, when monitoring adherent cells, oblique grazing incidence illumination can be used to enhance cell edge contrast; when observing suspended aggregates, vertical uniform diffuse illumination can be switched to avoid shadow occlusion. Simultaneously, the automatic locking function prevents illumination angle drift caused by wave-like swaying vibrations, ensuring consistent illumination conditions during long-term monitoring.

[0059] In some implementations, combined Figure 6 and Figure 8 As shown, the lighting module 2 is slidably connected to the top of the mounting groove 11 along its width direction. Specifically, a straight groove 14 is formed on the top of the mounting groove 11 along its width direction. A slider 24 matching the groove 14 is fixedly connected to the top of the ball socket 231. The slider 24 is embedded in the groove 14 and can move horizontally along the length direction of the groove 14. Optionally, guide limiting ribs (not shown in the figure) are provided on both sides of the groove 14, and corresponding mating grooves (not shown in the figure) are provided on both sides of the slider 24 to limit the vertical jump of the ball socket 231 while allowing it to slide smoothly along the width direction of the mounting groove 11. Preferably, the groove 14 is provided with elastic positioning protrusions or magnetic positioning components (not shown in the figure), which can automatically lock into place when the ball socket 231 slides to a preset position to prevent unexpected displacement due to vibration.

[0060] By configuring the lighting module 2 to slide along the width of the mounting slot 11 and achieve multi-angle adjustment in conjunction with the ball joint 23, the system can flexibly adjust the projection area of ​​the illumination spot on the bag wall according to the actual insertion position of the cell culture bag 200, the lateral offset of the bag body 201, or different bag sizes (such as width differences), ensuring that the near-wall target corresponding to the monitoring window 12 is always in the center of the optimal illumination field of view. Simultaneously, the lateral sliding function effectively compensates for the center offset of the bag body 201 caused by manufacturing tolerances of the disposable cell culture bag 200 or the bagging operation, ensuring that the illumination spot always accurately covers the monitoring window 12 area corresponding to the transparent carrier plate 13. For example, when using wider or narrower disposable cell culture bags 200, there is no need to replace the monitoring system hardware and keep the mounting base 1 against the side wall of the shaker 100; simply sliding the lighting module 2 laterally and fine-tuning the angle of the ball joint 23 is sufficient to quickly align with the target area, significantly improving the versatility and operational efficiency of the equipment. In addition, this sliding-rotation composite adjustment mechanism avoids problems such as edge illuminance attenuation, local shadows or imaging eccentricity caused by fixed lighting, ensuring the spatial consistency and temporal stability of lighting conditions during long-term dynamic monitoring.

[0061] In some implementations, such as Figure 5 and Figure 7 As shown, the monitoring system also includes a support plate 15. The support plate 15 is fixedly connected to the mounting base 1 near the... Figure 1 One side of the cell culture bag 200 shown is used to support and guide the disposable cell culture bag 200 to fit smoothly into the mounting groove 11 area. Specifically, the bottom surface of the support plate 15 is coplanar with the bottom surface of the mounting base 1, ensuring that the overall structure is placed stably on the shaker 100 platform; the top surface of the support plate 15 slopes downward in the direction away from the mounting base 1, forming a gentle slope guide surface, and smoothly connects with the bottom of the mounting groove 11, avoiding local wrinkles, suspension or stress concentration of the bag body 201 due to steps, edges or abrupt edges during loading or wave-like oscillation, thereby reducing mechanical disturbance to the flow field and cell distribution inside the bag.

[0062] Preferably, such as Figure 5 and Figure 9 As shown, transition plates 16 are fixedly connected to both sides of the support plate 15 along the length of the mounting groove 11. The top surface of the transition plate 16 slopes downward away from the support plate 15 and smoothly transitions with the top surface of the support plate 15, thus forming a continuous and uninterrupted guiding support surface in both the horizontal and vertical dimensions. This structure not only adapts to cell culture bags 200 of different widths, allowing the guide bag body 201 to naturally spread and closely fit the bottom of the transparent carrier plate 13 and the mounting groove 11, but also significantly increases the contact area between the monitoring system and the bottom of the cell culture bag 200, thereby pressing and fixing the monitoring system within the shaker 100 through the cell culture bag 200.

[0063] Furthermore, the bottom of the mounting base 1 and / or the support plate 15 is provided with an anti-slip pad (not shown in the figure), for example, on the bottom of the mounting base 1, or the bottom of the support plate 15, or both. The anti-slip pad is made of silicone, polyurethane, or medical-grade rubber material, which has a high coefficient of friction and good elasticity, and can effectively suppress the slippage or jumping of the monitoring system relative to the platform of the shaking table 100 during the periodic shaking of the shaking table 100, ensuring that the monitoring window 12 is always aligned with the target area.

[0064] The beneficial effects of this design are as follows: Firstly, the smooth and continuous support surface effectively avoids fluid disturbances caused by local suspension or frictional abrupt changes during dynamic rocking of the bag body 201, protecting adherent cells or sensitive aggregates from shear impact. Secondly, the expanded contact area allows the weight of the cell culture bag 200 to be applied more evenly to the monitoring system, improving the overall structural stability on the shaker 100 platform through the gravity compression effect. Combined with the bottom anti-slip pad, this significantly suppresses slippage, tilting, or micro-vibration of the monitoring system during prolonged wave motion. Thus, without introducing an additional puncture fixation mechanism, it ensures a mild cell culture environment while enhancing the mechanical robustness and optical alignment reliability of the monitoring device, fully meeting the comprehensive requirements of disposable bioreactors for non-invasive, highly stable online monitoring.

[0065] In some implementations, such as Figure 2 As shown, the monitoring system also includes a clamping component 3. The clamping component 3 is disposed at the end of the mounting base 1 and is used to detachably clamp and fix the mounting base 1 to the sealing strip 202 of the cell culture bag 200, thereby enabling the monitoring system to be quickly positioned and securely installed without damaging the sterile barrier.

[0066] Specifically, the clamping assembly 3 includes a connector 31 and a U-shaped clamp 32. The U-shaped clamp 32 is fixedly connected to the mounting base 1 via the connector 31, that is, one end of the connector 31 is fixedly connected to the end of the mounting base 1, and the other end of the connector 31 is fixedly connected to the U-shaped clamp 32. The opening of the U-shaped clamp 32 faces the sealing strip 202, and the opening size of the U-shaped clamp 32 matches the thickness of the sealing strip 202, so that the sealing strip 202 can be tightly embedded in the opening of the U-shaped clamp 32, forming a reliable mechanical clamping.

[0067] Preferably, such as Figure 3 As shown, an anti-slip strip 33 is provided on the inner wall of the U-shaped clamp 32. The anti-slip strip 33 is made of an elastic material with a high coefficient of friction (such as silicone, polyurethane or rubber) to increase the static friction of the clamping interface and prevent the monitoring system from loosening or shifting during the periodic shaking of the shaker 100.

[0068] Furthermore, the U-shaped clip 32 is slidably connected to the connector 31 in the vertical direction. Specifically, in conjunction with... Figure 3and Figure 4 As shown, the connector 31 includes a horizontal member 311 and a vertical member 312. The horizontal member 311 is horizontally positioned, with one end fixedly connected to the end of the mounting base 1 and the other end fixedly connected to the middle of the vertical member 312. The vertical member 312 is vertically positioned, with its bottom surface coplanar with the bottom surface of the mounting base 1 and able to contact the bottom of the shaker 100, providing additional support and stability. The vertical member 312 has a guide groove 313 along its length direction (i.e., the vertical direction). A guide block 321 is fixedly connected to one end of the U-shaped clamp 32 near the vertical member 312. The guide block 321 is embedded in the guide groove 313 and can slide up and down along the guide groove 313, thereby realizing the vertical sliding connection of the U-shaped clamp 32 relative to the connector 31. Through this structure, the U-shaped clamp 32 can be adjusted up and down according to the actual height or installation position of the sealing strip 202, improving its adaptability to different bioreactor models or assembly tolerances.

[0069] Preferably, a damping structure (not shown in the figure) is provided between the guide block 321 and the guide groove 313, such as an elastic silicone pad, a spring with friction plate, or a micro-tooth positioning mechanism. This damping structure can keep the U-shaped clamp 32 stable at any adjustable height without additional locking operation, preventing accidental slippage due to vibration or gravity, while allowing the user to manually apply force to smoothly adjust the position.

[0070] This solution utilizes a non-invasive clamping mechanism between the U-shaped clip 32 and the sealing tape 202, eliminating the need for perforation, bonding, or modification of the disposable cell culture bag 200 or the reactor body. This allows for rapid installation and disassembly of the monitoring system, maintaining its complete sealing and aseptic integrity. While ensuring clamping reliability, the U-shaped clip 32 is equipped with adaptive height adjustment capabilities, effectively accommodating sealing tapes 202 of varying thicknesses or installation heights. The coplanar design of the vertical component 312 and the bottom surface of the mounting base 1 provides auxiliary support, distributing some of the load and enhancing overall structural rigidity. The built-in damping structure achieves "stepless locking," balancing adjustment flexibility with operational stability, making it suitable for online monitoring needs under long-term, high-frequency oscillation conditions.

[0071] like Figure 7 and Figure 9 As shown, the mounting base 1 has a mounting cavity 17 inside, and the monitoring window 12 is connected to the mounting cavity 17. Inside the mounting cavity 17, along the optical path, are arranged the following components in sequence: optical path refraction component 4, imaging objective lens 5, and imaging module 6.

[0072] The optical path refraction assembly 4 includes at least one optical path refraction element 40 for deflecting a vertically incident light beam passing through the monitoring window 12 into a horizontal plane. The optical path refraction element 40 is preferably a right-angle prism or a plane mirror.

[0073] For example, the optical path refraction assembly 4 includes an optical path refraction element 40. Specifically, the optical path refraction element 40 is fixedly installed in the mounting cavity 17 directly below the monitoring window 12, and is configured to receive the imaging beam emitted from the target near the wall region of the cell culture bag 200 and passing vertically through the transparent carrier plate 13 and the monitoring window 12, and to bend it at a 90° angle to propagate it in the horizontal plane, so that the optical path extends laterally along the mounting base 1 to adapt to the layout of subsequent optical elements.

[0074] Preferably, the optical path refraction assembly 4 includes a plurality of optical path refraction elements 40 for refraction of the vertically incident light beam passing through the monitoring window 12 into a beam that propagates along the length of the mounting groove 11 in the horizontal plane. For example, the optical path refraction assembly 4 includes three optical path refraction elements 40, namely a first refraction element 41, a second refraction element 42 and a third refraction element 43, which are sequentially arranged in the mounting cavity 17 along the optical path.

[0075] Specifically, the first refractive element 41 is fixedly installed directly below the monitoring window 12, configured to receive the imaging beam emitted vertically downward from the transparent carrier plate 13 and deflect it by 90°, allowing the beam to propagate horizontally along the width of the mounting groove 11. The second refractive element 42 is fixedly installed in the output light path of the first refractive element 41, used to deflect the horizontal beam by another 90°, making it propagate vertically upward. The third refractive element 43 is located above the second refractive element 42 and aligned with it in the vertical direction, used to deflect the vertically upward beam by a third 90°, ultimately allowing it to propagate horizontally along the length of the mounting groove 11 and precisely collinear with the optical axis of the imaging objective lens 5.

[0076] Optionally, the first refractive element 41, the second refractive element 42 and the third refractive element 43 are all high-flatness optical plane mirrors with a wide-band high-reflectivity film (such as Al+SiO2 or Ag protective film) on their surfaces, and the reflection efficiency in the visible light band is not less than 95%.

[0077] The effect of this three-stage optical path deflection structure is that, within the wave-shaped bioreactor shaker 100, where both height and lateral space are limited, three 90° reflections (vertical-lateral-vertical-longitudinal) flexibly guide the imaging optical path from the originally vertically emitted near-wall region to the main optical axis extending along the length of the mounting groove 11, effectively avoiding interference from the sliding mechanism of the illumination module 2, the sidewalls of the mounting groove 11, and other mechanical structures. Simultaneously, this layout provides ample longitudinal mounting depth for the high numerical aperture imaging objective lens 5 and the imaging module 6, significantly improving system integration density and optical performance. Furthermore, the final optical path is arranged along the length of the bioreactor, consistent with the direction of the shaking motion, which helps reduce optomechanical disturbances caused by fluid sloshing, ensuring image stability, resolution, and repeatability during long-term online microscopic monitoring, meeting the requirements of non-invasive, high-reliability process analysis technology under GMP conditions.

[0078] like Figure 9 As shown, the imaging objective 5 is located on the outgoing light path of the optical path refraction assembly 4, and is used to perform high-resolution magnified imaging of targets in the near-wall region and generate corresponding optical images. Specifically, the imaging objective 5 is precisely mounted on the horizontal optical axis after the final bend of the optical path refraction assembly 4, and is precisely aligned with this optical axis to ensure that the imaging beam emitted from the optical path refraction assembly 4 can directly enter the imaging objective 5 for magnified imaging. The imaging objective 5 adopts a high numerical aperture (NA) design to achieve fine detail capture of near-wall microstructures or biological samples, while ensuring sufficient depth of field to cover the small undulations of the sample surface.

[0079] Optionally, the imaging objective 5 is equipped with a replaceable objective lens group (not shown in the figure) at its front end, including objectives with different magnifications (such as 10×, 20×, 40×, etc.) to meet diverse imaging needs and resolution requirements. These objectives are made of high-quality optical glass with a special coating treatment on the surface, exhibiting high transmittance and low chromatic aberration in the visible light range, thereby ensuring optimal image quality.

[0080] like Figure 9 As shown, the imaging module 6, electrically connected to the control module, is used to receive the optical image generated by the imaging objective 5 and output a digital image signal to the control module. Specifically, the imaging module 6 may employ existing imaging devices such as industrial cameras, scientific-grade sCMOS cameras, or embedded vision modules. These devices are image acquisition equipment known in the art, possessing the standard function of converting optical images into digital image signals and outputting them externally. They can be directly integrated into the mounting cavity 17 and aligned with the mirror surface of the imaging objective 5.

[0081] Among them, industrial cameras are suitable for conventional high frame rate and high stability monitoring scenarios; scientific-grade sCMOS cameras have low noise, high quantum efficiency and wide dynamic range, and are suitable for high sensitivity requirements such as low light or fluorescence imaging; embedded vision modules are small in size and low in power consumption, making them easy to be compactly laid out in space-constrained mounting brackets 1, and supporting edge preprocessing functions.

[0082] By selecting mature and standardized existing imaging devices as imaging module 6, high-reliability and high-compatibility image acquisition can be achieved without the need for additional dedicated hardware development, significantly reducing system integration difficulty and cost. Simultaneously, all of the aforementioned imaging devices support standard communication protocols (such as USB3 Vision, GigE Vision, etc.), enabling seamless interface with the control module and ensuring real-time and stable transmission of digital image signals, meeting the requirements of online bioreactor monitoring for imaging quality, long-term operational stability, and GMP compliance.

[0083] In some embodiments, the monitoring system also includes a relay lens group 7. The relay lens group 7 is disposed in the optical path within the mounting cavity 17 and is used to relay the optical image formed by the target in the near-wall region to maintain image quality and adapt to complex optical path layouts.

[0084] For example, the relay lens group 7 is located between two adjacent optical path refraction elements 40 of the optical path refraction assembly 4. Located as... Figure 7 The first refractive element 41 and the second refractive element 42 are located between, or between, the second refractive element 42 and the third refractive element 43. This scheme can perform wavefront correction and image refocusing on a beam that has traveled a certain distance after it has completed one 90° turn and before it enters the next turn. It effectively suppresses beam divergence, edge illumination attenuation, and high-frequency information loss caused by free space propagation. It is especially suitable for situations where the distance between two adjacent optical path refractive elements 40 is large (such as more than 50 mm), ensuring that the subsequent optical path receives a clear and collimated intermediate image.

[0085] For example, the relay lens group 7 is located between the exit end of the optical path refraction assembly 4 and the imaging objective lens 5, or between the imaging objective lens 5 and the imaging module 6. When located between the optical path refraction assembly 4 and the imaging objective lens 5, the relay lens group 7 re-images the intermediate image after multiple refractions onto the effective object plane position of the imaging objective lens 5, compensating for focusing errors caused by optical path offset or image plane drift, and improving the imaging efficiency of the objective lens. When located between the imaging objective lens 5 and the imaging module 6, the relay lens group 7 constitutes a "4f" relay system, transmitting the real image formed by the objective lens over a long distance to the photosensitive surface of the image sensor in the imaging module 6, avoiding field clipping or resolution reduction caused by the sensor not being able to be in close contact with the rear focal plane of the objective lens.

[0086] Specifically, the relay lens group 7 includes at least one pair of conjugate imaging lenses, preferably an achromatic cemented doublet lens pair or a four-element apochromatic structure, wherein its object-side and image-side focal planes are conjugate with the preceding and following stage optical image planes, respectively. Preferably, each lens surface is coated with a broadband anti-reflection film, with a single-sided reflectivity of less than 0.5% in the 400-700 nm wavelength band.

[0087] This solution significantly improves the imaging fidelity and layout flexibility of the folding microscope system by introducing a relay lens group 7 at key optical path nodes. Regardless of whether the optical path is elongated or undergoes multiple bends due to mechanical limitations, or whether the imaging module 6 needs to be offset due to spatial interference, the relay structure can maintain high-resolution, low-distortion image transmission, ensuring accurate identification of micron-level features such as cell adhesion morphology and aggregate size. Simultaneously, this solution does not require increasing the volume of the mounting base 1, is compatible with existing disposable cell culture bags 200 and shakers 100 structures, and meets the requirements for non-invasive, highly stable, and GMP-compliant online biological process monitoring.

[0088] In some embodiments, the monitoring system further includes an optical filter device for improving imaging contrast and suppressing interference signals. Specifically, the optical filter device includes a polarizer (not shown) disposed in the light output path of the illumination module 2, and an analyzer (not shown) and a wavelength selective filter (not shown) disposed in the light path within the mounting cavity 17, located between the imaging objective lens 5 and the imaging module 6.

[0089] A polarizer is fixedly connected to the light outlet of the illumination module 2, configured to convert the unpolarized light emitted by the LED light source 22 into linearly polarized light and illuminate the near-wall region of the cell culture bag 200. An analyzer is positioned in the imaging optical path, with the polarizer and analyzer orthogonally arranged (i.e., cross-polarization arrangement), to suppress specular reflection from the membrane surface of the cell culture bag 200. A wavelength-selective filter, which can be a bandpass, long-pass, or short-pass filter, is used to select a specific spectral band according to imaging requirements, such as 470nm±10nm for enhancing nuclear staining contrast, or 525nm±20nm for detecting green fluorescent protein (GFP), and is installed behind the analyzer or integrated with it.

[0090] This scheme, through a cross-polarization configuration (orthogonal polarizer and analyzer), effectively suppresses strong specular reflection light generated on the surface of the 200 polymer membrane in the cell culture bag, significantly reducing background glare. This highlights diffuse reflection or scattering signals from near-wall cells or aggregates, improving image contrast and detail visibility. Simultaneously, wavelength-selective filters limit the imaging spectral range, reduce stray light interference, enhance the signal-to-noise ratio of specific labeled signals, or match the optical characteristics of different cell types. The synergistic effect of these two methods enables the system to achieve high-quality observation in bright-field mode without fluorescent labeling, while also being compatible with fluorescence imaging requirements. This enhances the adaptability and imaging robustness of the monitoring system in various culture scenarios, including adherent cells, suspended aggregates, and labeled cells, meeting the technical requirements for non-invasive, highly reliable online visual analysis in biopharmaceutical processes.

[0091] In some implementations, such as Figure 9 As shown, the monitoring system also includes a drive module 8, and an imaging module 6 is connected to the output of the drive module 8. The drive module 8 is electrically connected to the control module and is used to drive the imaging module 6 to move along the optical axis of the imaging objective lens 5 to adjust the imaging distance between the imaging module 6 and the target in the near-wall region, thereby achieving focusing or adapting to different subsequent disposable cell culture bags 200.

[0092] Specifically, in combination Figure 9 and Figure 10As shown, the drive module 8 includes a drive motor 81 and a drive screw 82. The drive motor 81 is fixedly connected inside the mounting cavity 17, and the axis of its output shaft is parallel to the optical axis of the imaging objective lens 5. The screw of the drive screw 82 is coaxially fixedly connected to the output shaft of the drive motor 81 via a coupling. A drive block 83 is fixedly connected to the nut of the drive screw 82, and the imaging module 6 is fixedly connected to the top of the drive block 83. In use, the drive motor 81 drives the drive block 83 to move along the optical axis of the imaging objective lens 5 via the drive screw 82, thereby driving the imaging module 6 to move along the optical axis of the imaging objective lens 5.

[0093] This solution uses the drive module 8 to finely adjust the position of the imaging module 6 along the optical axis, dynamically compensating for surface shifts caused by manufacturing tolerances of the cell culture bag 200, differences in bag tightness, or changes in liquid level. This allows for automatic or manual focusing, ensuring that the target near the wall area remains within the optimal depth of field of the imaging objective lens 5. Especially during long-term continuous monitoring, this function effectively addresses focus drift caused by dynamic factors such as culture medium evaporation and bag deformation, ensuring image clarity and analytical accuracy. Furthermore, this structure eliminates the need to move the illumination module 2, the transparent carrier plate 13, or the culture bag itself, avoiding the introduction of additional disturbances and meeting the core requirements of non-invasive, sterile, closed bioreactor monitoring.

[0094] In some implementations, such as Figure 10 As shown, the monitoring system also includes a guide component 84. The guide component 84 is arranged along the optical axis of the imaging objective lens 5 and is used to guide and limit the movement of the imaging module 6, ensuring that it runs smoothly along a preset straight path during focusing and avoiding swaying, tilting or jamming.

[0095] Specifically, the guide assembly 84 includes a pair of parallel linear guide rails 841 and guide rail blocks 842 slidably mounted on the linear guide rails 841. The length direction of the linear guide rails 841 is parallel to the optical axis of the imaging objective lens 5, and the drive block 83 is fixedly connected to the top of the two guide rail blocks 842. Preferably, the linear guide rails 841 adopt a cross roller guide rail or micro ball guide rail structure, which has high rigidity, low coefficient of friction, and micron-level motion repeatability. Limiting blocks 843 are fixedly connected to both ends of the linear guide rails 841 to limit the travel range of the imaging module 6 and prevent it from exceeding its travel range and impacting the optical elements or leaving the optical path.

[0096] This solution, by setting a high-precision guide component 84, effectively constrains the imaging module 6 to translate only along the optical axis, significantly improving the straightness and stability of the focusing motion and avoiding optical axis misalignment, image blurring, or mechanical wear caused by lateral forces or assembly errors. Especially when used in conjunction with the drive module 8 for autofocus, the guide component 84 ensures that the photosensitive surface of the imaging sensor of the imaging module 6 is always perpendicular to the optical axis, maintaining optimal image quality. In addition, the limit stop 843 ensures the safety and reliability of system operation, making it suitable for long-term, unattended online monitoring scenarios of wave-type bioreactors, while meeting the engineering requirements of high integration and high robustness within a compact space.

[0097] This invention, without compromising the sterile seal of the disposable cell culture bag 200, achieves high-resolution, long-term, dynamic online microscopic monitoring of targets (such as adherent cells, microaggregates, etc.) in the near-wall region within the disposable cell culture bag 200 in a wave-shaped bioreactor through a synergistic design of mechanical positioning, controllable illumination, folded optical path imaging, and automatic image acquisition. Specifically, the mounting base 1 stably guides and fixes the cell culture bag 200 above the monitoring window 12 via the support plate 15, transition plate 16, and clamping assembly 3; the illumination module 2 provides uniform illumination with an adjustable angle from the top; the imaging beam passes vertically through the transparent carrier plate 13, undergoes multiple bends by the optical path refraction assembly 4 before entering the horizontal optical path, and is magnified and imaged by the imaging objective lens 5; the relay lens group 7, optical filter device, and drive focusing mechanism jointly ensure image quality and system adaptability; finally, the imaging module 6 converts the optical image into a digital signal and transmits it to the control module for analysis and processing.

[0098] The technical effects of this invention are as follows: (1) Non-invasive and sterile: No need to puncture or modify the cell culture bag 200, all contact materials meet the biocompatibility requirements, and the closedness of the disposable system is fully maintained; (2) High imaging quality and strong stability: Through high numerical aperture objective lens, cross polarization filter, relay imaging and autofocus mechanism, the membrane glare is effectively suppressed, the focus drift is compensated, and the micron-level cell structure is clearly captured; (3) Good versatility and environmental adaptability: The support and guidance structure, height adjustable clamping component 3 and multi-degree-of-freedom lighting module 2 can be adapted to disposable cell culture bags 200 of different specifications and wave-type bioreactor shaker 100; (4) Strong resistance to dynamic disturbance: Anti-slip pad, bottom coplanar support, damping lock and guide limit structure together ensure that the system operates stably for a long time under wave swing conditions; (5) Meets the requirements of process analysis technology (PAT): Supports real-time, quantitative and visual monitoring of key parameters such as cell adhesion, aggregate formation and biomass evolution, providing reliable data support for biopharmaceutical process control.

[0099] In summary, this invention provides a monitoring system that is compact, easy to operate, provides reliable imaging, and complies with GMP standards. It is particularly suitable for online process monitoring of disposable wave-type bioreactors and has significant engineering application value and industrialization prospects.

[0100] Example 2: This example provides a monitoring system for a bioreactor. The difference between this example and Example 1 is that the monitoring system further includes a phase sensor, which is electrically connected to the control module and is used to detect the motion phase signal of the bioreactor in real time, and output the zero phase reference point and the motion period T.

[0101] Specifically, the phase sensor is installed on the drive mechanism or swing shaft of the wave-type bioreactor shaker 100 to sense the periodic swinging motion of the shaker 100. The zero-phase reference point corresponds to the lowest point of the shaker 100's swing on the system mounting side, that is, the instant when the cell culture bag 200 swings to its lowest position with the shaker 100 and is about to reverse and rise; at this position, the bag's movement speed approaches zero, and the control module uses this as the phase timing reference. The motion period T is determined by the rotational speed of the bioreactor, with a typical rotational speed of 15 rpm, corresponding to a motion period T = 60 / 15 = 4 s.

[0102] The phase sensor can be implemented using any of the following types: (1) Rotary encoder: incremental photoelectric encoder or magnetoelectric encoder, whose code disk is coaxially installed with the shaft of the drive motor 81 of the rocker 100 or the swing spindle. The real-time angular position is obtained through orthogonal decoding, and the Z-phase signal outputs a zero-position pulse per revolution. After mechanical zero-point calibration, it corresponds to the lowest swing point of the rocker 100. (2) Hall sensor: cooperates with the permanent magnet fixed on the moving part of the rocker 100. When the rocker 100 swings past the lowest point, the magnet sweeps across the sensitive area of ​​the Hall element, and the output switch level jumps, generating a zero-phase reference pulse. (3) Magnetoresistive sensor: based on the anisotropic magnetoresistive (AMR) or giant magnetoresistive (GMR) effect, it detects the position change of the permanent magnet and outputs an analog voltage or digital pulse related to the swing angle. After threshold comparison, the zero-phase signal corresponding to the lowest point is extracted. (4) Inductive proximity switch: cooperates with the metal part of the rocker 100. When the rocker 100 swings to the lowest point, it detects the distance change and outputs a switch quantity zero-position pulse. (5) Accelerometer: The MEMS triaxial accelerometer is attached to the table of the rocker 100. The tilt angle of the rocker 100 is calculated by real-time acquisition of the gravitational acceleration components. The peak detection algorithm identifies the endpoint of the swing stroke and extracts the zero-phase reference point. (6) Laser rangefinder: Fixed to the base of the rocker 100, it emits a laser beam to the moving parts of the rocker 100 and receives the reflected signal. It measures the distance change in real time and identifies the position of the lowest point through waveform characteristics.

[0103] The control module calculates the current motion cycle T in real time based on the time interval between two adjacent zero-phase reference pulses; alternatively, it can directly use the user-set rotational speed value of 15 rpm and fix T=4s for phase delay calculation.

[0104] The control module is configured to execute the following phase-locked stroboscopic imaging and phase optimization procedure:

[0105] 1. Phase Scanning and Phase-Locked Trigger Acquisition: Within a preset phase scanning window, phase-locked trigger signals are generated sequentially according to a preset phase step, driving the illumination module 2 to emit short pulse light and simultaneously triggering the imaging module 6 to acquire image frames. Specifically, the control module automatically enters phase scanning mode upon system startup, replacement of the cell culture bag 200, or receipt of a user manual command. The preset phase scanning window covers two motion cycles, i.e., 2T = 8s, to ensure full capture of the complete characteristics of the periodic motion of the shaker 100 within the entire phase range, avoiding sampling deviations caused by accidental disturbances within a single cycle. The preset phase step Δφ ranges from 5° to 15° electrical angle, corresponding to a time step of Δt. step =(T / 360)×Δφ. Taking a motion period T=4000ms and a phase step of 10° as an example, the time step is 111.1ms. At each phase step position, the control module uses the current zero-phase reference point t0 as a reference, and calculates the time step based on the delay t corresponding to the phase angle φ. delay =(φ / 360)×T generates a single phase-locked loop trigger pulse. This trigger pulse is a TTL level signal with a rising edge effective and a pulse width of 10~50μs, which is sent to both the illumination module 2 and the imaging module 6. The illumination module 2 integrates a high-side MOSFET switching circuit, which instantaneously turns on the LED light source 22 after responding to the trigger signal, emitting a high-brightness short pulse light with a pulse width of 50~500μs; the imaging module 6 uses a global shutter CMOS sensor, which starts exposure on the rising edge of the trigger signal. The exposure time is preset to match the illumination pulse width (typical value 100~300μs), ensuring that light is only captured during the pulse illumination, effectively suppressing ambient stray light and motion blur.

[0106] 2. Calculation of stillness index: For each phase angle φ, at least two consecutive image frames are acquired to obtain the corresponding pixel matrix; the pixel values ​​of adjacent two frames are subtracted point by point, and the median absolute difference is calculated as the stillness index E(φ) for that phase, i.e.: Where φ represents the phase angle, median represents the median operation, and I t with I t+1E(φ) represents the pixel values ​​of two adjacent frames; the smaller the E(φ) value, the smaller the image change between adjacent frames, and the more static the image appears. Specifically, at the same phase angle φ, the control module continuously outputs two identical trigger pulses with a pulse interval not less than the sensor frame readout time (typically 10~30ms), thus obtaining two adjacent frame images I. t with I t+1 To further improve the statistical robustness of stillness assessment, three consecutive images can be acquired at the same phase angle. The average of the first two and last two images is calculated separately, or the minimum of the pairwise differences among the three images is taken as E(φ) for that phase. The control module reads the complete RAW format pixel matrix of two images, performs point-by-point subtraction within the FPGA or ARM core to generate a difference matrix, takes the absolute value of all pixels in the difference matrix, sorts the absolute value sequence, and extracts the median. Compared to the arithmetic mean, the median calculation is less sensitive to isolated large differences in a single image caused by microbubbles passing by, suspended particles shifting instantaneously, or random readout noise, ensuring that E(φ) truly reflects the overall motion stillness of the bag, culture medium, and adherent cell layer at that phase.

[0107] 3. Ideal Imaging Phase Recognition and Delay Calculation: Based on the above calculation results, the phase angle with the smallest E(φ) is selected as the ideal imaging phase φ. * According to the ideal imaging phase φ * The timing relationship between the phase angle and the zero-phase reference point is used to calculate the trigger delay Δt. Specifically, the control module iterates through all sampled phase angles, establishes a phase angle-static index mapping array, identifies the minimum value of E(φ) using bubble sort or quicksort algorithm, and records its corresponding phase angle as the ideal imaging phase φ. * 。 This phase angle represents the instantaneous moment within one motion cycle when the shaking speed of the shaker 100 is at its lowest, the tangential velocity of the near-wall region relative to the cell culture bag 200 approaches zero, and the imaging target is in its most stable state. Due to the nonlinear coupling effects of mechanical friction, inertial hysteresis, and liquid sloshing within the bag in the bioreactor's motion mechanism, φ is typically located at a phase offset after the lowest point of the shaker 100's swing (zero-phase reference point), and this offset fluctuates with changes in rotational speed, bag shape, and culture volume. This embodiment does not require any pre-set kinematic model and is entirely driven by measured image data for adaptive optimization. The control module uses the formula Δt = (φ...) * The trigger delay is calculated as ( / 360)×T, where φ * The time delay is measured in degrees, and T represents the current measured motion period (typically 4000 ms). This delay value represents the length of time the control system needs to wait until the ideal imaging phase from the zero-phase reference time t0.

[0108] 4. Synchronous Triggered Imaging: During subsequent imaging, the control module synchronously outputs a camera trigger signal to the imaging module and a strobe control signal to the illumination module at intervals of Δt after the zero-phase time t0 of each motion cycle. Specifically, after completing phase scanning and delay calculation, the system exits the scanning mode and automatically switches to the conventional online monitoring mode. In this mode, the control module receives the zero-position reference pulse output by the phase sensor in each motion cycle, using it as the reference at time t0, and immediately starts the hardware timer or DSP core timing unit. When the timer count reaches the trigger delay Δt calculated in step 3, the control module generates a set of synchronous trigger pulses through the GPIO port or a dedicated trigger output channel: the first trigger signal is sent to the imaging module 6, causing it to enter the exposure acquisition state; the second trigger signal is sent to the illumination module 2, causing it to emit short pulse illumination light precisely synchronized with the exposure window. The illumination pulse width is set in conjunction with the exposure time of the imaging module 6, with a typical value of 100~300μs, ensuring that "frozen" imaging is completed within the instantaneous window when the cell culture bag 200 has the lowest movement speed. This synchronous triggering mechanism is strictly repeated once within each motion cycle, thereby obtaining a series of phase angles strictly locked at φ during the culture process, which lasts for several hours to several weeks. * This embodiment produces a high-quality near-wall microscopic image sequence with excellent inter-frame spatiotemporal comparability. By introducing a phase sensor and the aforementioned control module configuration, the monitoring system possesses phase sensing and adaptive phase-locked imaging capabilities for the periodic motion of a wave-shaped bioreactor. Under typical operating conditions of 15 rpm and a cycle of 4 seconds, it can complete phase scanning in two motion cycles, automatically locking the ideal imaging phase window after the lowest point of the shaker's oscillation. This effectively solves the motion blur and imaging time drift problems caused by the continuous oscillation of the cell culture bag 200 in non-invasive near-wall monitoring under dynamic conditions, significantly improving the image clarity, data consistency, and process comparability of long-term online monitoring.

[0109] In some embodiments, the control module is further configured to automatically re-execute the phase scanning window to update the ideal imaging phase φ when a change in the rotational speed of the bioreactor is detected to exceed a preset threshold. * And its corresponding trigger delay Δt. Specifically, in the normal online monitoring mode, the control module continuously monitors the zero-phase reference pulse output by the phase sensor, calculates the time interval between adjacent pulses in real time, and thus obtains the measured value T of the current motion cycle. actual When T actual The motion period T calibrated relative to the previous scan cycle calibratedWhen the rate of change exceeds a preset threshold (preferably ±5%), or when the deviation between the user-set rotational speed and the currently measured rotational speed exceeds the corresponding threshold, the control module determines that the reactor's motion state has changed significantly and automatically triggers a re-entry into the phase scanning mode. The system re-executes the complete dual-cycle phase scanning, stationarity index calculation, and ideal imaging phase optimization process according to steps 1 to 3 to obtain the updated ideal imaging phase φ. * new and trigger delay Δt new This is used to replace the original parameters for subsequent imaging triggering.

[0110] This adaptive update mechanism ensures that even when the rotation speed drifts due to process adjustments, load changes, or equipment aging during cultivation, the monitoring system can still automatically lock the optimal imaging phase window without manual intervention or shutdown for calibration, thus maintaining the clarity, stability, and batch comparability of near-wall region imaging over the long term.

[0111] In some implementations, the control module is further configured to: limit the calculation area of ​​the stillness index E(φ) to the image sub-region corresponding to the near-wall target, and perform masking or median filtering anti-interference algorithm when foam interference is detected.

[0112] Specifically, the near-wall target corresponds to a thin optical layer of 0-300 μm thickness near the inner wall of the cell culture bag 200. This region is the core observation zone for cell adhesion, aggregate formation, and biomass dynamics. Before calculating the stillness index E(φ), the control module first extracts a sub-image matrix covering only the projection area of ​​this near-wall thin layer from the complete pixel matrix based on the preset region of interest coordinates. Subsequent point-by-point subtraction and median calculations are all limited to this sub-region. This region limitation strategy effectively eliminates the interference of suspended cell populations far from the bag wall, turbulent areas of the culture medium, and irrelevant structures deep within the bag on the stillness assessment, ensuring that E(φ) truly reflects the degree of motion stillness of the near-wall target itself. Meanwhile, the control module integrates a foam interference recognition and suppression algorithm in the image preprocessing stage: when a bright patch or striped reflective halo caused by bubble bursting or surfactant enrichment is detected in a sub-region of the image, the system automatically generates a binary mask to exclude the pixel region affected by foam interference from the difference calculation; or an adaptive median filtering algorithm is used to replace the original pixel value with the median gray value in the neighborhood of the interfered pixel, so as to suppress the bright noise of foam while preserving the edge details of cells and aggregates.

[0113] This anti-interference mechanism significantly improves the imaging robustness and data reliability of the monitoring system under conditions of serum-containing culture medium, high-density fermentation, and intense aeration.

[0114] Example 3: This example provides a monitoring method for a bioreactor, based on the monitoring system for a bioreactor described in Example 2, including the following steps: S1. Acquire the motion phase signal of the bioreactor and determine the zero-phase reference point and motion period T. S2. Within a preset phase scanning window, generate phase-locked trigger signals sequentially according to a preset phase step, driving the illumination module 2 to emit short pulse light and simultaneously triggering the imaging module 6 to acquire image frames. S3. For each phase angle φ, continuously acquire at least two image frames to obtain the corresponding pixel matrix; calculate the median absolute difference by subtracting the pixel values ​​of adjacent two image frames point by point, which serves as the stillness index E(φ) for that phase, i.e.: Where φ represents the phase angle, median represents the median operation, and I t with I t+1 E(φ) represents the pixel values ​​of two adjacent frames; the smaller the value of E(φ), the smaller the image change between adjacent frames, and the more static the image appears. S4. Select the phase angle with the smallest E(φ) as the ideal imaging phase φ. * According to the ideal imaging phase φ * Calculate the trigger delay Δt based on the time relationship with the zero-phase reference point. S5. In each subsequent motion cycle, after the zero-phase time t0, at an interval of Δt, synchronously output the camera trigger signal to the imaging module 6 and output the strobe control signal to the illumination module 2.

[0115] In some embodiments, the method further includes the following step: S6, real-time monitoring of the motion cycle changes of the bioreactor; when a change in rotation speed is detected to exceed a preset threshold, steps S2 to S4 are automatically repeated to update the ideal imaging phase φ. * And its corresponding trigger delay Δt.

[0116] In some implementations, the following steps are also included: S7, limiting the calculation area of ​​the stillness index E(φ) to the image sub-region corresponding to the near-wall target, and performing masking or median filtering anti-interference algorithm when foam interference is detected.

[0117] The specific implementation methods, parameter ranges, preferred conditions, and control logic of the above steps are exactly the same as the description of the control module being configured to perform the corresponding functions in Embodiment 2.

[0118] Specifically, this includes: the zero-phase reference point corresponds to the lowest point of the shaker's oscillation on the system installation side; the motion period T is determined by the bioreactor's rotation speed, typically 15 rpm with a period T = 4 s; the phase scanning window covers two motion periods, i.e., 2T = 8 s; the phase sensor can be implemented using various types such as a rotary encoder, Hall sensor, magnetoresistive sensor, inductive proximity switch, accelerometer, or laser rangefinder; the stationarity index E(φ) is calculated using the median absolute difference and its noise reduction principle; the adaptive recognition algorithm for the ideal imaging phase φ; the calculation formula for the trigger delay Δt is Δt = (φ / 360) × T; the preset threshold for rotation speed change is ±5%; the near-wall target corresponds to the optical thin-film projection area with a thickness of 0~300 μm on the inner side of the bag wall; and the foam interference suppression algorithm includes a mask exclusion method and an adaptive median filtering method.

[0119] To save space in the specification and avoid unnecessary repetition with Embodiment 2, this embodiment will not elaborate on the above technical details one by one, and all of its contents are incorporated herein by reference.

[0120] This embodiment, by executing the above steps, realizes a phase-locked stroboscopic synchronous imaging, static index adaptive optimization, automatic recalibration for speed drift, and near-wall region anti-interference imaging method that is completely corresponding to the monitoring system described in Embodiment 2. It can complete the initial phase scan in two motion cycles under the dynamic swing conditions of a typical wave-type bioreactor with a speed of 15 rpm and a cycle of 4 s, and automatically respond to changes in speed and relock the ideal imaging phase during long-term culture. At the same time, it effectively suppresses the influence of non-target areas and foam interference on static assessment, thereby continuously acquiring high-definition, high spatiotemporal comparability, and high signal-to-noise ratio near-wall microscopic image sequences, providing reliable data support for long-term quantitative analysis of cell adhesion, aggregate formation, and biomass dynamic trends.

[0121] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0122] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes several instructions to cause a computer terminal (which may be a mobile phone, computer, server, or network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0123] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A monitoring system for a bioreactor, characterized in that, Used to monitor cell adhesion, aggregate formation and biomass dynamics in the near-wall region of a cell culture bag (200); the cell culture bag (200) includes a bag body (201) and sealing strips (202) connected to opposite sides of the bag body (201), the bag body (201) is used to contain cell culture medium and carry out cell culture, and the sealing strips (202) are used to fix it to the bioreactor; The monitoring system includes a control module and a mounting base (1). The mounting base (1) has a mounting groove (11) on one side for accommodating the side wall of the bag body (201). A monitoring window (12) is provided at the bottom of the mounting groove (11). A lighting module (2) is provided at the top of the mounting groove (11). The lighting module (2) is electrically connected to the control module and is used to illuminate the target in the near-wall area corresponding to the monitoring window (12). The mounting base (1) has an internal mounting cavity (17), and the monitoring window (12) communicates with the mounting cavity (17); the mounting cavity (17) is provided with the following components arranged sequentially along the optical path: The optical path refraction assembly (4) includes at least one optical path refraction element (40) for refraction of the vertically incident light beam passing through the monitoring window (12) into the horizontal plane for propagation. The imaging objective (5) is located on the outgoing light path of the optical path refraction assembly (4) and is used to perform high-resolution magnification imaging of the target in the near-wall area and generate a corresponding optical image. The imaging module (6), electrically connected to the control module, is used to receive the optical image generated by the imaging objective (5) and output a digital image signal to the control module; The monitoring system also includes a phase sensor, which is electrically connected to the control module and is used to detect the motion phase signal of the bioreactor in real time and output a zero-phase reference point and motion period T. The control module is configured as follows: Within the preset phase scanning window, phase-locked trigger signals are generated sequentially according to the preset phase step, driving the illumination module (2) to emit short pulse light and simultaneously triggering the imaging module (6) to acquire image frames; For each phase angle φ, at least two consecutive image frames are acquired to obtain the corresponding pixel matrix; the pixel values ​​of two adjacent frames are subtracted point by point, and the median absolute difference is calculated as the stillness index E(φ) for that phase, i.e.: ； Where φ represents the phase angle, median represents the median operation, and I t with I t+1 E(φ) represents the pixel value between two adjacent frames; the smaller the value, the smaller the change in the image between adjacent frames, and the more static the image is. Based on the above calculation results, the phase angle with the smallest E(φ) is selected as the ideal imaging phase φ*, and the trigger delay Δt is calculated according to the time relationship between the ideal imaging phase φ* and the zero phase reference point. During subsequent imaging, the control module outputs a camera trigger signal to the imaging module (6) and a strobe control signal to the illumination module (2) at intervals of Δt after the zero-phase time t0 of each motion cycle.

2. The monitoring system for a bioreactor according to claim 1, characterized in that, The optical path refraction assembly (4) includes a plurality of optical path refraction elements (40) for refraction of the vertically incident light beam passing through the monitoring window (12) into a beam that propagates along the length of the mounting groove (11) in the horizontal plane.

3. The monitoring system for a bioreactor according to claim 1, characterized in that, The monitoring system also includes a relay lens group (7), which is disposed in the optical path within the mounting cavity (17) for relaying the optical image formed by the target in the near-wall region.

4. A monitoring system for a bioreactor according to claim 1, characterized in that, The monitoring system also includes an optical filter device, which includes a polarizer disposed in the light output path of the illumination module (2), and an analyzer and a wavelength selective filter disposed in the light path within the mounting cavity (17) and located between the imaging objective (5) and the imaging module (6).

5. A monitoring system for a bioreactor according to claim 1, characterized in that, The monitoring system also includes a drive module (8), and the imaging module (6) is connected to the output end of the drive module (8); the drive module (8) is electrically connected to the control module and is used to drive the imaging module (6) to move along the optical axis of the imaging objective (5) to adjust the imaging distance between the imaging module (6) and the target in the near-wall area; The monitoring system further includes a guide component (84), which is arranged along the optical axis of the imaging objective (5) and is used to guide and limit the movement of the imaging module (6).

6. A monitoring system for a bioreactor according to claim 1, characterized in that, The monitoring system also includes a support plate (15), which is disposed on the side of the mounting base (1) near the cell culture bag (200); the bottom surface of the support plate (15) is coplanar with the bottom surface of the mounting base (1), and the top surface is inclined downward in a direction away from the mounting base (1) and smoothly connected to the bottom of the mounting groove (11). Among them, the support plate (15) is provided with transition plates (16) on both sides along the length direction of the mounting groove (11). The top surface of the transition plate (16) is inclined downward in the direction away from the support plate (15) and smoothly connected with the top surface of the support plate (15). And / or, the bottom of the mounting base (1) and / or the support plate (15) is provided with an anti-slip pad.

7. A monitoring system for a bioreactor according to claim 1, characterized in that, The monitoring system also includes a clamping component (3), which is disposed at the end of the mounting base (1) and is used to clamp and fix the mounting base (1) on the sealing strip (202); The clamping assembly (3) includes a connector (31) and a U-shaped clamp (32). The U-shaped clamp (32) is connected to the mounting base (1) through the connector (31), and the opening size of the U-shaped clamp (32) matches the thickness of the sealing strip (202). The inner wall of the U-shaped clip (32) is provided with anti-slip strips (33). And / or, the U-shaped clip (32) is slidably connected to the connector (31) in the vertical direction.

8. A monitoring system for a bioreactor according to claim 1, characterized in that, A transparent carrier plate (13) is provided inside the monitoring window (12), and the top surface of the transparent carrier plate (13) is coplanar with the bottom surface of the mounting groove (11); A transparent heating film is provided on the transparent carrier plate (13); And / or, a flexible attachment pad is provided on the inner wall of the mounting groove (11); And / or, the lighting module (2) is rotatably connected to the top of the mounting slot (11); And / or, the lighting module (2) is slidably connected to the top of the mounting groove (11) along the width direction of the mounting groove (11).

9. A monitoring method for a bioreactor, characterized in that, The implementation of the monitoring system for a bioreactor according to claim 1 includes the following steps: S1. Obtain the motion phase signal of the bioreactor and determine the zero-phase reference point and motion period T; S2. Within the preset phase scanning window, phase-locked trigger signals are generated sequentially according to the preset phase step to drive the illumination module (2) to emit short pulse light and simultaneously trigger the imaging module (6) to acquire image frames. S3. For each phase angle φ, continuously acquire at least two frames of images to obtain the corresponding pixel matrix; calculate the median absolute difference of the pixel values ​​of two adjacent frames as the stillness index E(φ) for that phase, i.e.: ； Where φ represents the phase angle, median represents the median operation, and I t with I t+1 E(φ) represents the pixel value between two adjacent frames; the smaller the value, the smaller the change in the image between adjacent frames, and the more static the image is. S4. Select the phase angle with the smallest E(φ) as the ideal imaging phase φ. * According to the ideal imaging phase φ * Calculate the trigger delay Δt based on the time relationship between the zero-phase reference point and the reference point. S5. In each subsequent motion cycle, after the zero phase time t0, at an interval of Δt, the camera trigger signal is synchronously output to the imaging module (6), and the strobe control signal is output to the illumination module (2).

Images