Three-dimensional microscopic system and method for tracking bacterial movement in real time

Abstract

The invention discloses a three-dimensional microscopic system and method for tracking bacterial movement in real time, and belongs to the technical field of microscop.The three-dimensional microscopic system comprises a microscope, a camera, a two-dimensional objective table, a piezoelectric positioning device and an upper computer; the microscope is used for carrying out microscopic imaging on the sample carried by the two-dimensional objective table, and the camera is used for acquiring a microscopic image of the sample after being imaged by the microscope; the two-dimensional objective table is used for driving a sample to perform two-dimensional planar motion; the piezoelectric positioning device is used for driving an objective lens of the microscope to move in the direction perpendicular to the two-dimensional plane; the upper computer is electrically connected with the camera, the two-dimensional objective table and the piezoelectric positioning device, and the upper computer is used for controlling the movement of the two-dimensional objective table and the piezoelectric positioning device so as to enable bacteria in the sample to be in a clear view of the camera in real time. The real-time position, speed, movement direction and other information of the bacteria in the three-dimensional space can be obtained, and therefore the three-dimensional movement law and response behaviors of the bacteria in different environments can be revealed.

Description

Technical Field

[0001] The present invention relates to the field of microscopy technology, and particularly relates to a three-dimensional microscopy system and method for real-time tracking of bacterial movement. Background Art

[0002] The movement behavior of bacteria is closely related to the occurrence and spread of various diseases. For example, the motility of Helicobacter pylori enables it to penetrate the gastric mucosa, leading to diseases such as gastric ulcers and gastric cancer. In addition, the motility of pathogenic bacteria also affects their efficiency of invading hosts, thereby affecting the spread rate of infections. In-depth study of the movement characteristics of bacteria helps to discover potential disease treatment targets.

[0003] Bacterial movement is one of the important characteristics of bacterial physiological activities. Different types of bacteria achieve movement through different ways, specifically including the following movement methods: 1. Flagella-driven swimming: Many bacteria rely on flagella for movement, such as Escherichia coli and Salmonella. Flagella are long spiral structures protruding outside bacterial cells, and there are motor-like structures at their bases, which push bacteria forward through rotational movement. The movement direction of flagella can be changed at any time, enabling bacteria to alternate forward in a "swimming - pausing" mode. A typical manifestation is the alternating behavior of bacteria "running" and "flipping" in fluids.

[0004] 2. Gliding: Some bacteria lack flagella but can achieve movement through surface gliding, such as some myxobacteria and green algae. Gliding movement usually occurs on solid surfaces, and bacteria slowly move on the surface through sticky substances secreted by the cell surface or other mechanisms.

[0005] 3. Clustering movement: Certain bacteria (such as streptococci) tend to aggregate into cluster-like groups for movement. By jointly secreting extracellular polymers, they move in a mutually dependent manner. This movement is usually seen in high-density bacterial populations.

[0006] 4. Tumble movement: A small number of bacteria have short cilia structures and perform rolling or rotational movements through the tumbling action of cilia or interaction with the environmental surface.

[0007] The movement of bacteria is affected by their internal and external environments. For example, the flagella of Escherichia coli and the rotation of the body during movement will change the direction of the surrounding fluid. The fluid flows towards the bacteria while the fluid at the head and tail ends moves away from the bacteria. Furthermore, an adsorption effect is generated when approaching the solid wall surface, causing the enrichment of bacteria on the surface. A series of properties such as chemical gradients (chemotaxis), the viscosity and rheological properties of the environmental fluid can all affect the movement characteristics of bacteria. Studying the movement behavior of bacteria is of great significance for understanding the physiological functions of microorganisms and their interactions with the environment.

[0008] In the prior art, optical microscopes and fluorescence microscopes are mainly used to record the movement of bacteria. In recent years, some technologies based on high-frame-rate cameras and confocal microscopes have also been developed. The main imaging methods include the following: 1. Two-dimensional optical microscopy imaging: Two-dimensional optical microscopy imaging is an imaging technology that uses an optical microscope to observe and record samples in a plane. This method mainly relies on visible light being focused onto the sample through an objective lens to form an image, and is widely used in fields such as biology and materials science to observe the planar structure and movement patterns of samples such as cells and bacteria. With its simple and intuitive imaging method, optical microscopes enable researchers to quickly understand the morphology and movement trajectories of samples in two-dimensional space.

[0009] 2. Fluorescence imaging: Fluorescence imaging is an imaging technology that uses fluorescent dyes or fluorescent proteins to label specific structures in a sample, and then observes their behavior and distribution under a microscope. The principle of fluorescence imaging is to excite fluorescent molecules with light of a specific wavelength, causing them to emit light of different wavelengths, and forming an image by detecting the emitted light. Fluorescence imaging technology is widely used in biomedical research, especially in the study of the molecular structure, dynamic behavior, and interactions of bacteria, cells, and tissues.

[0010] 3. Confocal microscopy imaging: Confocal microscopy imaging is an optical imaging technology that obtains three-dimensional structure information by scanning the sample layer by layer. Compared with traditional optical microscopes, confocal microscopes reduce background light interference by focusing on a specific plane of the sample, thereby generating clearer images. Its core principle is to use a pinhole to filter out non-focal-plane light signals and only retain the light from the focal plane, improving image resolution and contrast. A typical confocal microscope can also combine fluorescence labeling to make cells or bacteria emit light at a specific wavelength, enhancing the visibility of the target area.

[0011] However, there are some defects and deficiencies in the above-mentioned imaging methods in the prior art.

[0012] Firstly, it is impossible to track a single bacterium for a long time and over a large range. Optical and fluorescence microscopes mainly provide two-dimensional imaging with a fixed range. Therefore, when the bacterium swims beyond the imaging range of the microscope, its trajectory cannot be captured.

[0013] Secondly, depth information is missing. The actual movement of bacteria is three-dimensional. When the bacterium makes a vertical movement, it will cause defocusing in the imaging, and ultimately the bacterium's trajectory will be lost. Summary of the Invention

[0014] Aiming at the problems that existing imaging devices cannot track bacteria and lack depth information, the purpose of the present invention is to provide a three-dimensional microscopy system and method for real-time tracking of bacterial movement, so as to at least partially solve the above problems.

[0015] To achieve the above object, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a three-dimensional microscopy system for real-time tracking of bacterial movement, including a microscope, a camera, a two-dimensional stage, a piezoelectric positioning device, and a host computer; the microscope is used for microscopic imaging of a sample carried by the two-dimensional stage, the camera is used for obtaining a microscopic image of the sample after imaging by the microscope; the two-dimensional stage is used for driving the sample to perform two-dimensional planar movement; the piezoelectric positioning device is used for driving the objective lens of the microscope to move in a direction perpendicular to the two-dimensional plane; the host computer is electrically connected to the camera, the two-dimensional stage, and the piezoelectric positioning device, and the host computer is used for making the bacteria in the sample be in the clear field of view of the camera in real time by controlling the movement of the two-dimensional stage and the piezoelectric positioning device.

[0016] In some preferred embodiments, a fixture for clamping the sample is provided on the two-dimensional stage; the fixture includes a fixture body, a light-transmitting window is opened on the fixture body, two sliding members spanning the light-transmitting window are slidably connected to the fixture body, and pressing members for pressing the sample are arranged on the sliding members.

[0017] In some preferred embodiments, a groove for placing the sample is provided on the sliding member.

[0018] In some preferred embodiments, the pressing member is a shrapnel.

[0019] In some preferred embodiments, the sliding members are each provided with a locking member for fixing their positions.

[0020] In some preferred embodiments, the microscope is an inverted microscope, and the two-dimensional stage is located on one side above the microscope.

[0021] In some preferred embodiments, it further includes an independent controller electrically connected to the two-dimensional stage and an external handle electrically connected to the independent controller.

[0022] In a second aspect, the present invention further provides a three-dimensional microscopy method for real-time tracking of bacterial movement, the method is applied to the system as described above, and the method includes the following steps: The host computer obtains the current frame image of the sample from the camera and detects whether there are bacteria in it. If there are, it proceeds to the next step; if not, it continues to obtain and detect subsequent frame images. The host computer locks the real-time position of the bacteria in subsequent frames of images through a tracking algorithm, and analyzes the imaging characteristics of the bacteria in each frame of image during the tracking process; The host computer controls the movement of the two-dimensional stage and the piezoelectric positioning device according to the tracking and imaging characteristic analysis results of the bacteria, so that the bacteria in each frame of image are real-time in the clear field of view of the camera.

[0023] In some preferred embodiments, the tracking algorithm is the MOSSE algorithm.

[0024] In some preferred embodiments, the purpose of analyzing the imaging characteristics of the bacteria in the image is to judge whether the bacteria in the image appear defocused.

[0025] Adopting the above technical solutions, the beneficial effects of the present invention are as follows: 1. Overcome the limitation of two-dimensional imaging and realize the three-dimensional trajectory capture of bacteria. Through the setting of the two-dimensional stage and the piezoelectric positioning device, the two can move under the control of the host computer, so that the camera can comprehensively track the three-dimensional trajectory of the bacteria, obtain information such as the real-time position, speed and movement direction of the bacteria in three-dimensional space, and thus be able to reveal the three-dimensional movement law and response behavior of the bacteria in different environments.

[0026] 2. Solve the problem of fast movement tracking. The three-dimensional microscopy system provided by the present invention combines high-speed imaging and positioning algorithms, and can obtain the movement data of bacteria with extremely high time resolution, capture the minute behavior details such as their fast swimming, turning, aggregation, etc., so as to provide more accurate kinematic data of bacteria.

[0027] 3. Avoid the problems of phototoxicity and photobleaching and realize long-term tracking of live bacteria. Conventional fluorescence imaging or confocal microscopy is prone to cause phototoxicity when observing live bacteria for a long time, affecting the natural movement state of bacteria or causing photobleaching. Since the three-dimensional microscopy system provided by the present invention has high tracking efficiency for bacteria, it only needs to be equipped with a lower-intensity light source (low light conditions) to obtain clear images, thereby reducing the damage to live bacteria and realizing long-term dynamic observation of bacteria, which is helpful for studying the persistent adaptation behavior and group interaction of bacteria.

[0028] 4. Improve the spatial resolution and accurately reveal the movement patterns and interactions of bacteria. The three-dimensional microscopy system provided by the present invention makes the minute movements and interaction patterns of bacteria in a complex environment clearer through high-resolution imaging and precise motion detection techniques. For example, it can identify minute movement behaviors such as the rotation and tumbling of bacteria, and reveal the chemotactic response of bacteria in different concentration gradients, which is crucial for understanding the infection mechanism, group behavior and bacterial ecological relationship of pathogenic bacteria. Description of the Drawings

[0029] Figure 1 This is a schematic structural diagram of the present invention.

[0030] Figure 2 This is a schematic structural diagram of the fixture in the present invention.

[0031] In the figure: 1 - microscope, 2 - camera, 3 - two-dimensional stage, 4 - piezoelectric positioning device, 5 - host computer, 6 - fixture, 61 - fixture body, 62 - light-transmitting window, 63 - sliding member, 64 - slider, 65 - chute, 66 - locking member, 67 - pressing member. Specific embodiments

[0032] The following further describes the specific embodiments of the present invention with reference to the accompanying drawings. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Embodiment 1 A three-dimensional microscopic system for real-time tracking of bacterial movement, as Figure 1 shown, includes a microscope 1, a camera 2, a two-dimensional stage 3, a piezoelectric positioning device 4 and a host computer 5.

[0034] The microscope 1 is used for microscopic imaging of the sample carried by the two-dimensional stage 3. For example, the microscope 1 is configured as an inverted microscope, such as the Nikon Ti2-U inverted microscope. Correspondingly, the two-dimensional stage 3 is located on the upper side of the microscope 1, and the light source used for microscopy is located on the upper side of the two-dimensional stage 3.

[0035] The camera 2 is used to collect the microscopic images of the sample after being imaged by the microscope. The camera 2 is selected as a camera with high frame rate and high resolution, such as the PCO edge 5.5 sCMOS camera. The camera 2 is connected to the camera interface equipped with the microscope 1, so as to conveniently obtain microscopic images.

[0036] The two-dimensional stage 3 is used to drive the sample to perform two-dimensional planar movement. The two-dimensional stage 3 is configured as an electrically controllable electric stage, and it performs two-dimensional movement in the horizontal plane.

[0037] A notch for installing the fixture 6 is provided on the two-dimensional stage 3. The fixture 6 is used to clamp and fix the sample. The sample is usually placed on a glass slide. The sample is usually a liquid, colloid or solid material containing bacteria to be observed, and the fixture 6 is specifically used to clamp the glass slide.

[0038] As Figure 2As shown, the fixture 6 includes a fixture body 61. The fixture body 61 has a rectangular frame structure, and its four corners are fixed by screws in the notches (in a stepped shape) opened on the two-dimensional stage 3. The fixture body 61 has a light-transmitting window 62.

[0039] Two sliding members 63 spanning the light-transmitting window 62 are also slidably connected to the fixture body 61. For example, both sliding members 63 are strip-shaped and arranged along the width direction of the fixture body 61. Sliders 64 are fixed to both ends of the sliding member 63, and corresponding chutes 65 adapted to the sliders 64 are opened on the two side frames of the fixture body 61 along the length direction, so that the sliding member 63 can slide along the length direction. Usually, the sliding member 63 is also provided with a locking member 66 for fixing its position, such as a locking screw screwed on the slider, and the position locking function of the sliding member 63 is realized by making the end of the locking screw press against or away from the chute 65.

[0040] In addition, grooves for placing the glass slide are provided on the top surfaces of the opposite sides of the two sliding members 63. When the distance between the two sliding members 63 is appropriate, the glass slide can be just clamped between the grooves on the two sliding members 63. In addition, a pressing member 67 for pressing the glass slide is configured on each sliding member 63, such as a spring piece. One end of the spring piece is fixed to the sliding member 63 by screws or welding, and the other end of the spring piece applies a pressing force to the glass slide to fix the glass slide.

[0041] The piezoelectric positioning device 4 is used to drive the objective lens of the microscope 1 to move in a direction perpendicular to the two-dimensional plane. The piezoelectric positioning device 4 is configured as a piezoelectric scanner, such as a high-dynamic piezoelectric scanner, which is fixedly connected to the objective lens of the microscope 1 and is used to drive the objective lens of the microscope 1 to move in the vertical direction.

[0042] It is easy to understand that the movement directions of the two-dimensional stage 3 and the piezoelectric positioning device 4 depend on the actual arrangement state of the objective lens in the microscope 1. Specifically, the two-dimensional movement plane of the two-dimensional stage 3 is perpendicular to the objective lens, and the movement direction of the piezoelectric positioning device 4 is parallel to the objective lens. When the objective lens is arranged in the vertical direction, the two-dimensional stage 3 drives the sample to perform two-dimensional movement in the horizontal plane, and the piezoelectric positioning device 4 drives the objective lens to move in the vertical direction.

[0043] The host computer 5 is electrically connected to the camera 2, the two-dimensional stage 3, and the piezoelectric positioning device 4. For example, the host computer 5 has a PCIe 3.0 slot in which a capture card is installed to facilitate the acquisition of microscopic images obtained by the camera 2. The host computer 5 is specifically electrically connected to the two-dimensional stage 3 through the stage controller 7 and to the piezoelectric positioning device 4 through the piezoelectric scanner controller 8. In this way, the host computer 5 can control the movements of the two-dimensional stage 3 and the piezoelectric positioning device 4, so that the bacteria in the sample can present clear images thereof in real time, facilitating the observation by researchers.

[0044] Of course, the host computer 5 can also be only used for image display. Correspondingly, the two-dimensional stage 3 and the piezoelectric positioning device 4 are both electrically connected to independent controllers, and both are equipped with external handles electrically connected to their respective independent controllers. In this way, under the guidance of the images displayed by the host computer 5, researchers can manually operate the two-dimensional stage 3 and the piezoelectric positioning device 4, so that the bacteria in the sample can present clear images thereof in real time. And it is easy to understand that the above-mentioned stage controller 7 and piezoelectric scanner controller 8 can be used as independent controllers, enabling them to receive both the control instructions from the host computer 5 and the control instructions issued by the external handle.

[0045] Embodiment 2 A three-dimensional microscopy method for real-time tracking of bacterial movement, which is applied to the system disclosed in the above Embodiment 1. The method includes the following steps: The host computer obtains the current frame image of the sample from the camera and detects whether there are bacteria therein. If there are, it proceeds to the next step; if not, it continues to obtain and detect subsequent frame images. The host computer locks the real-time positions of the bacteria in subsequent frame images through a tracking algorithm and analyzes the imaging characteristics of the bacteria in each frame image during the tracking process. The host computer controls the movements of the two-dimensional stage and the piezoelectric positioning device according to the tracking and imaging characteristic analysis results of the bacteria, so that the bacteria in each frame image are in the clear field of view of the camera in real time.

[0046] Among them, the tracking algorithm is the MOSSE algorithm.

[0047] Among them, the purpose of analyzing the imaging characteristics of the bacteria in the image is to judge whether the bacteria in the image show defocus phenomenon.

[0048] When the host computer receives the stop condition, the host computer stops executing the above method. The stop condition is triggered, for example, by a set timing program or by a peripheral device connected to the host computer.

[0049] It should be noted that in the description of the present invention, the orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "front", "rear", etc. is an illustration of the structure of the present invention based on the drawings shown. It is only for the convenience of describing the present invention simply, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation to the present invention.

[0050] Regarding "first" and "second" in this technical solution, they are only used to distinguish the names of the same or similar structures, or corresponding structures with similar functions, rather than arranging the importance of these structures, nor having any meaning of ranking, comparing sizes, or the like.

[0051] In addition, unless otherwise clearly specified and limited, the terms "installation" and "connection" should be understood in a broad sense. For example, the connection can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two structures. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the general idea of the present invention and in connection with the specific context of this solution.

[0052] The embodiments of the present invention have been described in detail above in conjunction with the drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. A three-dimensional microscopic system for real-time tracking of bacterial movement, characterized in that: It includes a microscope, a camera, a two-dimensional stage, a piezoelectric positioning device and a host computer; the microscope is used for microscopically imaging the sample carried by the two-dimensional stage, and the camera is used for acquiring the microscopic image of the sample after being imaged by the microscope; the two-dimensional stage is used for driving the sample to perform two-dimensional planar movement; the piezoelectric positioning device is used for driving the objective lens of the microscope to move in a direction perpendicular to the two-dimensional plane; the host computer is electrically connected to the camera, the two-dimensional stage and the piezoelectric positioning device, and the host computer is used for making the bacteria in the sample be in the clear field of view of the camera in real time by controlling the movement of the two-dimensional stage and the piezoelectric positioning device.

2. The three-dimensional microscopic system according to claim 1, characterized in that: A fixture for clamping the sample is provided on the two-dimensional stage; the fixture includes a fixture body, a light-transmitting window is opened on the fixture body, and two sliding members straddling the light-transmitting window are slidably connected to the fixture body, and pressing members for pressing the sample are arranged on the sliding members.

3. The three-dimensional microscopic system according to claim 2, wherein: A groove for placing the sample is provided on the sliding member.

4. The three-dimensional microscopic system according to claim 2, wherein: The pressing member is a spring piece.

5. The three-dimensional microscopic system according to claim 2, wherein: The sliding members are all provided with locking members for fixing their positions.

6. The three-dimensional microscopic system according to claim 1, characterized in that: The microscope is an inverted microscope, and the two-dimensional stage is located on one side above the microscope.

7. The three-dimensional microscopic system according to claim 1, wherein: It further includes an independent controller electrically connected to the two-dimensional stage and an external handle electrically connected to the independent controller.

8. A three-dimensional microscopy method for real-time tracking of bacterial movement, the method being applied to the system according to any one of claims 1-7, characterized in that: The method includes the following steps: The host computer acquires the current frame image of the sample from the camera and detects whether there are bacteria in it. If there are, it proceeds to the next step; if not, it continues to acquire and detect subsequent frame images. The host computer locks the real-time position of the bacteria in subsequent frame images through a tracking algorithm, and analyzes the imaging characteristics of the bacteria in each frame image during the tracking process. Based on the tracking and imaging characteristic analysis results of the bacteria, the host computer controls the movement of the two-dimensional stage and the piezoelectric positioning device, so that the bacteria in each frame image are in the clear field of view of the camera in real time.

9. The method according to claim 8, characterized in that: The tracking algorithm is the MOSSE algorithm.

10. The method according to claim 8, wherein: The purpose of analyzing the imaging characteristics of the bacteria in the image is to judge whether the bacteria in the image appear defocused.

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