Continuous ultra-thin section observation integrated equipment for hard tissue sample and use method of continuous ultra-thin section observation integrated equipment

Abstract

The invention discloses continuous ultra-thin section observation integrated equipment for a hard tissue sample and a use method, and belongs to the technical field of histomicrotomes, and the specific scheme is that the integrated equipment comprises a base, a first reciprocating guide rail, a second reciprocating guide rail, a feeding guide rail, an air static pressure main shaft, a vacuum chuck, a cutter system and a camera system, the first reciprocating guide rail and the feeding guide rail are perpendicular to each other and are fixed to the base, the axis of the air static pressure main shaft is perpendicular to the first reciprocating guide rail, the air static pressure main shaft reciprocates along the first reciprocating guide rail, the vacuum suction cup is fixed to the front end of the air static pressure main shaft, a sample is fixed to the vacuum suction cup, and the tool system and the sample are oppositely arranged. The cutter system and the camera system reciprocate along the second reciprocating guide rail; the second reciprocating guide rail is parallel to the first reciprocating guide rail, the second reciprocating guide rail is fixed to the base, and the base reciprocates along the feeding guide rail. Automatic preparation and layer-by-layer observation of the multilayer ultrathin sections can be continuously and stably completed, and the pathological diagnosis time is greatly shortened.

Description

Technical Field

[0001] The present invention belongs to the technical field of microtomes, and particularly relates to an integrated device and method for continuously observing ultra-thin sections of hard tissue samples. Background Art

[0002] As the "gold standard" for pathological diagnosis, the diagnostic accuracy of tissue sectioning technology is directly related to the sectioning accuracy. Due to the poor conformal quality of traditional paraffin embedding technology, the prepared sections have low accuracy and it is difficult to clearly observe the subcellular structural features. In contrast, resin embedding technology significantly improves the conformal accuracy of tissue samples, but the hard and brittle characteristics of its materials pose higher requirements for sectioning equipment. Traditional sectioning equipment, due to the lack of precise control ability for moving parts, is prone to problems such as unstable section quality and uneven thickness when cutting resin-embedded samples. Especially when the section thickness reaches the sub-micron level, the samples are extremely prone to fragmentation due to mechanical stress, seriously restricting the visualization and analysis of subcellular pathological features.

[0003] In recent years, the three-dimensional reconstruction technology of tissue sections has provided a supplementary dimension for pathological diagnosis, improving the comprehensiveness and accuracy of diagnosis. However, there are still many bottlenecks in this technical process: before observing each sample section, it is necessary to go through cumbersome pretreatment steps such as anti-detachment, staining, and dehydration, which are time-consuming and prone to sample detachment and loss. In addition, during the three-dimensional reconstruction of multiple consecutive sections, problems such as displacement deviation and alignment difficulties between sections lead to a significant decrease in the accuracy of three-dimensional reconstruction, further restricting the reliability of the diagnostic results. If "in-situ observation" of samples can be achieved during sectioning, the above problems can be effectively solved.

[0004] Therefore, there is an urgent need to develop a new device that can achieve high-precision ultra-thin continuous sectioning of hard tissues. This device needs to have the ability to stably cut hard tissue samples, ensure that the section thickness reaches the sub-micron level without fragmentation, and at the same time integrate the function of in-situ observation of samples, so as to achieve precise alignment and data integration during three-dimensional reconstruction. The breakthrough of this device will provide core hardware support for pathological diagnosis, promote the leapfrog development of clinical diagnosis from two-dimensional observation to high-precision three-dimensional analysis, and ultimately improve the sensitivity and specificity of disease diagnosis. Summary of the Invention

[0005] The main problem solved by the present invention is that hard tissue sectioning is limited by the accuracy and processing methods of traditional sectioning equipment, and it is impossible to achieve complete and continuous sub-micron sections; the secondary problem is that traditional tissue three-dimensional reconstruction faces problems such as cumbersome sample section pretreatment steps, excessive displacement deviation between layers, and difficult alignment, resulting in a decrease in three-dimensional reconstruction accuracy. To solve the above technical problems, the present invention provides an integrated device and method for continuously observing ultra-thin sections of hard tissue samples.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An integrated equipment for continuously observing ultra-thin sections of hard tissue samples, comprising a base, a first reciprocating guide rail, a second reciprocating guide rail, a feed guide rail, an air static pressure spindle, a vacuum chuck, a tool system and a camera system. The first reciprocating guide rail and the feed guide rail are perpendicular to each other and are both fixed on the base. The axis of the air static pressure spindle is perpendicular to the first reciprocating guide rail. The spindle seat of the air static pressure spindle together with the air static pressure spindle reciprocates along the first reciprocating guide rail. The vacuum chuck is fixed at the front end of the air static pressure spindle. During sectioning, the sample is adsorbed on the vacuum chuck. The tool system is arranged opposite to the sample. Both the tool system and the camera system reciprocate along the second reciprocating guide rail. The second reciprocating guide rail is parallel to the first reciprocating guide rail. The second reciprocating guide rail is fixed on the base, and the base reciprocates along the feed guide rail.

[0008] Further, the tool system includes a tool, a tool holder and a tool holder seat. The tool is arranged opposite to the sample. The tool is fixed on the tool holder. The tool holder is fixed on the tool holder seat. The tool holder seat reciprocates along the second reciprocating guide rail.

[0009] Preferably, the camera system is fixed on the tool holder seat.

[0010] Further, the vacuum chuck is connected to a vacuum system.

[0011] Further, the height of the sample is the same as the height of the tool.

[0012] Further, the integrated equipment further includes a displacement sensor. The displacement sensor is assembled on the air static pressure spindle and is used to detect the coaxiality deviation between the rotation axis of the sample and the vacuum chuck.

[0013] Further, the air static pressure spindle is internally provided with a dynamic balance detector, and a number of threaded holes are arranged at the circumference of the vacuum chuck.

[0014] Preferably, the tool is a diamond tool.

[0015] Further, the height of the lens in the camera system is the same as the height of the sample.

[0016] Further, the integrated equipment further includes a control system, a first driving device, a second driving device, and a third driving device. The first driving device drives the spindle seat of the aerostatic spindle together with the aerostatic spindle to move along the first reciprocating guide rail. The second driving device drives the tool holder system and the camera system to move along the second reciprocating guide rail. The third driving device drives the base to move along the feed guide rail. The aerostatic spindle, the vacuum system, the camera system, the first driving device, the second driving device, and the third driving device are all electrically connected and / or signal-connected to the control system.

[0017] Preferably, the first driving device, the second driving device, and the third driving device are all servo motors.

[0018] The aerostatic spindle 5 is driven by a motor to rotate.

[0019] A method for using the integrated equipment for continuously observing ultra-thin sections of hard tissue samples includes the following steps:

[0020] Step 1: Adsorb the hard tissue sample to be cut on the surface of the vacuum chuck, ensuring that the coaxiality error between the rotation axis of the sample and the vacuum chuck is ≤1 μm; start the motor to drive the aerostatic spindle to rotate counterclockwise at a speed of 500 - 3000 rpm, and the dynamic unbalance of the aerostatic spindle is stabilized within the range of ≤0.5 mg.

[0021] Step 2: Control the tool system to move along the second reciprocating guide rail and move the tool to the front of the sample; control the base to drive the tool to approach the surface of the sample along the feed guide rail at a speed of 1 - 5 mm / min until micro-cutting is triggered to complete the initial contact calibration between the tool and the sample.

[0022] Step 3: Control the spindle seat of the aerostatic spindle to drive the sample to reciprocate along the first reciprocating guide rail at a speed of 5 - 30 mm / min. At the same time, control the base to continuously advance the tool towards the sample along the feed guide rail at a cutting depth of 5 - 20 μm, and gradually remove the surface material of the sample through layer-by-layer rough cutting until the surface of the sample is evenly involved in cutting to form a flat initial cutting surface.

[0023] Step 4: Switch to the precision cutting mode, control the base to continuously advance the tool towards the sample along the feed guide rail at a cutting depth of 0.1 - 1 μm, and at the same time control the spindle seat of the aerostatic spindle to drive the sample to reciprocate along the first reciprocating guide rail at a speed of 1 - 10 mm / min.

[0024] Step 5: After completing the single-layer cutting, control the tool to quickly retract to a safe position, and move the sample to the observation area of the camera system along the first reciprocating guide rail by controlling the spindle seat of the aerostatic spindle to image the surface of the sample, and save the image data in real time and automatically number it.

[0025] Step 6: Repeat Step 4 - Step 5, continuously cut and observe the surface of the sample until the slicing of the preset number of layers is completed. The image data of the slices is automatically stored in the system database in chronological order, providing high-precision spatial coordinates and topography information for subsequent 3D reconstruction.

[0026] In Step 1, a displacement sensor is used to detect the coaxiality deviation between the sample and the vacuum chuck, and the position of the sample is adjusted to ensure that the coaxiality error between its rotation axis and the vacuum chuck is ≤1μm. The dynamic unbalance of the aerostatic spindle is measured in real time by an internal dynamic balance detector. According to the detection results, dynamic balance blocks are gradually added to the preset threaded holes on the circumference of the vacuum chuck until the dynamic unbalance of the aerostatic spindle is stabilized within the range of ≤0.5mg.

[0027] An integrated equipment for continuous ultra-thin section observation of hard tissue samples proposed by the present invention effectively solves the core problems of easy fragmentation, low precision, and observation disconnection in traditional sectioning equipment during sub-micron sectioning by innovatively combining ultra-precision machine tool structures, high-precision motion control, and online detection technologies. The specific beneficial effects are mainly reflected in the following aspects:

[0028] (1) By the coordinated control of an ultra-precision guide rail and a diamond tool, the single-layer cutting thickness of the sample reaches the sub-micron level, and the integrity of the slices is significantly improved, solving the technical bottleneck of easy fragmentation in current hard tissue ultra-thin sectioning and providing hardware guarantee for the visualization of subcellular pathological features.

[0029] (2) An integrated real-time online observation device is integrated, which can synchronously obtain the surface information of the sample during the sectioning process, avoiding the displacement and alignment deviation in the observation of traditional continuous sections, and improving the 3D reconstruction accuracy by more than 50%.

[0030] (3) Through numerical control programming, the equipment can continuously and stably complete the automated preparation and layer-by-layer observation of multiple ultra-thin sections. The single-section observation efficiency is increased to 1 section per minute (thickness ≤1μm), significantly reducing the dependence on manual operation and greatly shortening the pathological diagnosis time. Description of the Drawings

[0031] Figure 1 is the axonometric drawing of the present invention;

[0032] In the figure, 1, base; 2, first reciprocating guide rail; 3, second reciprocating guide rail; 4, feed guide rail; 5, aerostatic spindle; 6, vacuum chuck; 7, tool system; 8, camera system; 9, sample; 10, base; 71, tool; 72, tool holder; 73, tool holder base. Detailed Embodiments

[0033] The technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0034] Embodiment 1:

[0035] The specific implementation method of the hard tissue sample continuous ultra-thin section observation equipment of the present invention is as follows:

[0036] Step 1: Adsorb the hard tissue sample to be cut on the surface of the vacuum chuck 6, and achieve the close fit between the sample 9 and the vacuum chuck 6 through the vacuum system. Use a high-precision displacement sensor (resolution ≤ 0.1μm) to detect the coaxiality deviation between the sample 9 and the vacuum chuck 6, and ensure that the coaxiality error between its rotation axis and the vacuum chuck 6 is ≤ 1μm by finely adjusting the position of the sample 9.

[0037] Step 2: Start the aerostatic spindle 5 and rotate it counterclockwise at a speed of 1000 rpm. Measure the dynamic unbalance of the aerostatic spindle 5 in real time through the built-in dynamic balance detector. According to the detection results, gradually add dynamic balance blocks into the preset threaded holes on the circumference of the vacuum chuck 6 until the dynamic unbalance of the aerostatic spindle 5 is stabilized within the range of ≤ 0.5 mg.

[0038] Step 3: Control the tool system 7 to move along the second reciprocating guide rail 3, and move the diamond tool to the front of the sample 9. Subsequently, control the base 10 to drive the tool 71 to slowly approach the surface of the sample 9 at a speed of 1 mm / min along the feed guide rail 4 until micro-cutting is triggered to complete the initial contact calibration between the tool 71 and the sample 9.

[0039] Step 4: Control the spindle seat of the aerostatic spindle 5 to drive the sample 9 to reciprocate along the first reciprocating guide rail 2 at a speed of 10 mm / min, and at the same time control the base 10 to continuously advance the tool towards the sample 9 along the feed guide rail 4 with a cutting depth of 10μm. In this stage, the surface material of the sample 9 is gradually removed by layer-by-layer rough cutting until the surface of the sample 9 is uniformly involved in cutting to form a flat initial cutting surface.

[0040] Step 5: Switch to the precision cutting mode, control the base 10 to continuously advance the tool towards the sample 9 along the feed guide rail 4 with a cutting depth of 0.5μm, and at the same time control the spindle seat of the aerostatic spindle 5 to drive the sample to slowly move along the first reciprocating guide rail 2 at a speed of 3 mm / min.

[0041] Step 6: After completing the single-layer cutting, control the tool 71 to quickly retract to a safe position (5 mm away from the sample), and move the sample to the observation area of the integrated camera system 8 along the first reciprocating guide 2 by controlling the aerostatic spindle 5. Image the surface of the sample using a high-resolution microscopic camera, save the image data in real time, and automatically number it.

[0042] Step 7: Repeat Steps 5-6, continuously cut and observe the surface of the sample until 100 preset layers of slices are completed (total cutting depth of 50 μm). The image data of each layer of slices is automatically stored in the system database in chronological order, providing high-precision spatial coordinates and topography information for subsequent three-dimensional reconstruction.

[0043] Step 8: After the cutting is completed, control the diamond tool to retract to the initial position, stop the rotation of the aerostatic spindle 5, and release the sample by the vacuum chuck 6.

[0044] The present invention is different from the traditional slicing form. The present invention first removes the surface material of the sample and then takes images of the sample surface to obtain continuous multiple layers of sample surface images with a layer spacing ≤ 1 μm.

[0045] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An integrated equipment for continuously observing ultra-thin sections of hard tissue samples, characterized in that: It includes a base (1), a first reciprocating guide rail (2), a second reciprocating guide rail (3), a feed guide rail (4), an air static pressure spindle (5), a vacuum chuck (6), a tool system (7) and a camera system (8). The first reciprocating guide rail (2) and the feed guide rail (4) are perpendicular to each other and are both fixed on the base (1). The axis of the air static pressure spindle (5) is perpendicular to the first reciprocating guide rail (2). The spindle seat of the air static pressure spindle (5) reciprocates along the first reciprocating guide rail (2). The vacuum chuck (6) is fixed at the front end of the air static pressure spindle (5), and a sample (9) is adsorbed on the vacuum chuck (6). The tool system (7) is arranged opposite to the sample (9). Both the tool system (7) and the camera system (8) reciprocate along the second reciprocating guide rail (3). The second reciprocating guide rail (3) is parallel to the first reciprocating guide rail (2), and the second reciprocating guide rail (3) is fixed on a base (10), and the base (10) reciprocates along the feed guide rail (4).

2. The integrated equipment for continuous ultra-thin section observation of hard tissue samples according to claim 1, characterized in that: The tool system (7) includes a tool (71), a tool holder (72) and a tool holder seat (73). The tool (71) is arranged opposite to the sample (9) and has the same height. The tool (71) is fixed on the tool holder (72), the tool holder (72) is fixed on the tool holder seat (73), and the tool holder seat (73) reciprocates along the second reciprocating guide rail (3). The camera system (8) is fixed on the tool holder seat (73), and the height of its lens is the same as the height of the sample (9).

3. The integrated equipment for continuous ultra-thin section observation of hard tissue samples according to claim 1, characterized in that: The vacuum chuck (6) is connected to a vacuum system.

4. An integrated equipment for continuously observing ultra-thin sections of hard tissue samples according to claim 1, characterized in that: The integrated device further includes a displacement sensor, and the displacement sensor is assembled on the air static pressure spindle (5) for detecting the coaxiality deviation between the rotation axis of the sample (9) and the vacuum chuck (6).

5. The integrated equipment for continuously observing ultra-thin sections of hard tissue samples according to claim 1, characterized in that: The air static pressure spindle (5) is internally provided with a dynamic balance detector, and several threaded holes are provided at the circumference of the vacuum chuck (6).

6. The integrated equipment for continuous ultra-thin section observation of hard tissue samples according to claim 2, wherein: The tool (71) is a diamond tool.

7. An integrated equipment for continuous ultra-thin section observation of hard tissue samples according to claim 3, characterized in that: The integrated equipment further includes a control system, a first driving device, a second driving device and a third driving device. The first driving device drives the spindle seat of the air static pressure spindle (5) to move along the first reciprocating guide rail (2). The second driving device drives the tool system (7) and the camera system (8) to move along the second reciprocating guide rail (3). The third driving device drives the base (10) to move along the feed guide rail (4). The air static pressure spindle (5), the vacuum system, the camera system (8), the first driving device, the second driving device and the third driving device are all electrically connected and / or signal-connected to the control system.

8. A method for using an integrated equipment for continuously observing ultra-thin sections of a hard tissue sample according to any one of claims 2-7, characterized in that, It includes the following steps: Step 1: Adsorb the hard tissue sample (9) to be cut on the surface of the vacuum chuck (6) to ensure that the coaxiality error between the rotation axis of the sample (9) and the vacuum chuck (6) ≤ 1 μm. Start the motor to drive the air static pressure spindle (5) to rotate counterclockwise, and the dynamic unbalance of the air static pressure spindle (5) is stabilized within the range of ≤ 0.5 mg. Step 2: Control the tool system (7) to move along the second reciprocating guide rail (3) and move the tool (71) to the front of the sample (9); control the base (10) to drive the tool (71) to approach the surface of the sample (9) along the feed guide rail (4) at a speed of 1 - 5 mm / min until micro-cutting is triggered, and complete the initial contact calibration between the tool (71) and the sample (9). Step 3: Control the seat of the aerostatic spindle (5) to drive the sample (9) to reciprocate along the first reciprocating guide rail (2) at a speed of 5 - 30 mm / min. At the same time, control the base (10) to continuously advance the tool (71) towards the sample (9) along the feed guide rail (4) with a cutting depth of 5 - 20 μm, and gradually remove the surface material of the sample (9) through layer-by-layer rough cutting until the surface of the sample (9) is evenly involved in cutting to form a flat initial cutting surface; Step 4: Switch to the precision cutting mode, control the base (10) to continuously advance the tool (71) towards the sample (9) along the feed guide rail (4) with a cutting depth of 0.1 - 1 μm. At the same time, control the seat of the aerostatic spindle (5) to drive the sample (9) to reciprocate along the first reciprocating guide rail (2) at a speed of 1 - 10 mm / min; Step 5: After completing single-layer cutting, control the tool (71) to quickly retract to a safe position, and move the sample (9) to the observation area of the camera system (8) along the first reciprocating guide rail (2) by controlling the seat of the aerostatic spindle (5) to image the surface of the sample (9), and save the image data in real time and automatically number it; Step 6: Repeat Steps 4 - 5, continuously cut and observe the surface of the sample (9) until the preset number of slices is completed. The image data of the slices is automatically stored in the system database in chronological order to provide high-precision spatial coordinates and topography information for subsequent 3D reconstruction.

9. The method of use according to claim 8, characterized in that: In Step 1, use a displacement sensor to detect the coaxiality deviation between the sample (9) and the vacuum chuck (6), and adjust the position of the sample (9) to ensure that the coaxiality error between its rotation axis and the vacuum chuck (6) is ≤1 μm; use a built-in dynamic balance detector to measure the dynamic unbalance of the aerostatic spindle (5) in real time. According to the detection results, gradually add dynamic balance blocks in the preset threaded holes on the circumference of the vacuum chuck (6) until the dynamic unbalance of the aerostatic spindle (5) is stabilized within the range of ≤0.5 mg.

10. The method of use according to claim 8, characterized in that: In Step 2, the rotational speed of the aerostatic spindle is 500 - 3000 rpm.

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