CN112326652B

CN112326652B - Imaging method, device and system for controlling sequence determination reaction

Info

Publication number: CN112326652B
Application number: CN201710855394.2A
Authority: CN
Inventors: 周志良; 郑焦; 徐家宏; 徐剑峰; 王光明; 姜泽飞; 颜钦
Original assignee: Genemind Biosciences Co Ltd
Current assignee: Genemind Biosciences Co Ltd
Priority date: 2017-09-20
Filing date: 2017-09-20
Publication date: 2021-09-03
Anticipated expiration: 2037-09-20
Also published as: WO2019056834A1; CN112326652A

Abstract

The invention discloses an imaging method, a device and a system for controlling sequence determination reaction. The imaging method is used for an optical detection system, the optical detection system comprises an imaging device and a bearing device, the bearing device comprises a temperature control device and a carrying platform, the imaging device comprises a lens module, the lens module comprises an optical axis, the carrying platform is used for bearing a sample, the temperature control device is used for adjusting the temperature of the sample, and the imaging method comprises the following steps: before the imaging device is used for carrying out image acquisition on the sample or when the imaging device is used for carrying out image acquisition on the sample, the temperature control device is used for setting the range of temperature fluctuation of the sample, so that the position fluctuation range of the lens module along the optical axis is located in the preset range. Therefore, by using the imaging method, the position fluctuation range of the lens module can be controlled within a preset range, and adverse effects on the imaging device during image acquisition are reduced or avoided.

Description

Imaging method, device and system for controlling sequence determination reaction

Technical Field

The present invention relates to the field of optical detection, and in particular, to an imaging method, a method for controlling a sequence determination reaction, a control device, an optical detection system, and a sequence determination system.

Background

Sequencing, i.e., sequencing, includes determination of nucleic acid sequences. Sequencing platforms on the market at present comprise a first-generation sequencing platform, a second-generation sequencing platform and a third-generation sequencing platform. From a functional control perspective, the sequencing instrument includes a detection module that is used to translate and/or collect changes in information generated by biochemical reactions in the sequencing to determine the sequence. The detection module generally includes an optical detection module, a current detection module, and an acid-base (pH) detection module. The sequencing platform based on the optical detection principle performs sequence determination by analyzing and collecting detected optical signal changes in sequencing biochemical reactions.

In the process of collecting optical signals, the lens module of the optical detection module may generate unexpected position fluctuation, and the position fluctuation brings adverse effect on the collection of the optical signals.

Disclosure of Invention

Embodiments of the present invention are directed to solving at least one of the technical problems occurring in the related art or at least providing an alternative practical solution. To this end, embodiments of the present invention need to provide an imaging method, a method of controlling a sequencing reaction, a control device, an optical detection system, and a sequencing system.

The inventors have created the idea of making the solution of the invention based on the following findings:

those skilled in the art can know that, in the imaging, image/signal acquisition or sequencing process, especially in a platform including a high-precision/high-power optical system, if a lens module (including an objective lens) of an optical detection system shakes/fluctuates or the shake/fluctuation is not controlled, it is likely to affect the image acquisition and the signal acquisition.

When an imaging device is used to focus a reaction device (e.g., a chip) on a sequencing platform, the inventors surprisingly found that a variation curve (Z-x) of coordinates (defined as coordinates in the Z-axis direction) of a lens module (including an objective lens) along a focusing direction (e.g., the channel direction of the chip, defined as the x-direction) does not conform to a theoretical linear relationship or is approximately linear, exhibits a severe local fluctuation, and the fluctuation exhibits a periodic variation, as shown in fig. 1. Further, the inventor has tested that even if no focus tracking is performed, i.e., no command is given to the lens module to move in the Z-axis direction, the lens module only takes a picture of the same field of view, and the Z-axis direction of the lens module still fluctuates periodically, as shown in fig. 2, with a period of about 10.4 s. More surprisingly, the inventors found that the period of the fluctuation is very close to the period of the variation of the temperature of the reaction device, and suspected that the fluctuation of the lens module may be related to the fluctuation of the temperature. Generally, the setting for allowing the temperature variation of the reaction apparatus is controlled by a temperature control device connected to the reaction apparatus, for example, it is desirable that the temperature of the reaction apparatus is maintained at about 25 ℃, the temperature variation range is set to [24 ℃, 26 ℃ C ], that is, more than 25 ± 1 ℃ by the temperature control device, and the temperature control device adjusts the temperature so as to maintain the temperature of the reaction apparatus within the preset range.

The inventor verifies and verifies the guess by controlling the temperature fluctuation, for example, the temperature control device is removed, the behavior similar to focus tracking is carried out back and forth along one channel of the chip, the Z-X fluctuation of the lens module accords with the theoretical linear or approximately linear relation, and the above-mentioned periodic fluctuation phenomenon does not occur. Based on the above findings and verifications, the inventors propose a solution for controlling the lens module fluctuation by controlling the temperature variation.

An embodiment of the present invention provides an imaging method, where the imaging method is used in an optical detection system, the optical detection system includes an imaging device and a carrying device, the carrying device includes a temperature control device and a stage, the imaging device includes a lens module, the lens module includes an optical axis, the stage is used for carrying a sample, the temperature control device is used for adjusting the temperature of the sample, and the method includes:

before the imaging device is used for carrying out image acquisition on the sample or when the imaging device is used for carrying out image acquisition on the sample, the temperature control device is used for setting a range for allowing the temperature of the sample to fluctuate, so that the fluctuation range of the position of the lens module along the optical axis is within a preset range.

The embodiment of the invention provides a method for controlling a sequence determination reaction, which utilizes a sequence determination system to control the sequence determination reaction,

the sequence determination system comprises an optical detection system, the optical detection system comprises an imaging device and a bearing device, the imaging device comprises a lens module, the lens module comprises an optical axis, the bearing device comprises a temperature control device and a carrying platform, the carrying platform is used for carrying a sample, the sequence determination reaction comprises the step of carrying out image acquisition on the sample by using the imaging device, and the method comprises the following steps:

before the sequence determination reaction is performed by the sequence determination system or when the sequence determination is performed by the sequence determination system, setting a range in which the temperature fluctuation of the sample is allowed by the temperature control device so that a position fluctuation range of the lens module along the optical axis is within a preset range.

By using the imaging method and/or the sequence determination control method, the position fluctuation range of the lens module can be controlled within a preset range, and adverse effects on the imaging device during image acquisition are reduced or avoided.

An optical detection system of an embodiment of the present invention includes a control device, an imaging device, and a carrying device, where the carrying device includes a temperature control device and a stage, the imaging device includes a lens module, the lens module includes an optical axis, the stage is used for carrying a sample, the temperature control device is used for adjusting the temperature of the sample, and the control device is used for: before the imaging device is used for carrying out image acquisition on the sample or when the imaging device is used for carrying out image acquisition on the sample, the temperature control device is used for setting a range for allowing the temperature of the sample to fluctuate, so that the fluctuation range of the position of the lens module along the optical axis is within a preset range.

A sequence measurement system according to an embodiment of the present invention controls a sequence measurement reaction, and includes an optical detection system, where the optical detection system includes a control device, an imaging device, and a carrying device, the imaging device includes a lens module, the lens module includes an optical axis, the carrying device includes a temperature control device and a stage, the stage is used to carry a sample, and the control device is used to perform image acquisition on the sample by using the imaging device, and is used to:

By utilizing the optical detection system and/or the sequence determination system, the position fluctuation range of the lens module can be controlled within a preset range, and adverse effects on the imaging device during image acquisition are reduced or avoided.

A control device for controlling imaging according to an embodiment of the present invention is used in an optical detection system, where the optical detection system includes an imaging device and a carrying device, the carrying device includes a temperature control device and a stage, the imaging device includes a lens module, the lens module includes an optical axis, the stage is used for carrying a sample, the temperature control device is used for adjusting the temperature of the sample, and the control device includes:

a storage device for storing data, the data comprising a computer executable program;

a processor configured to execute the computer-executable program, wherein executing the computer-executable program comprises performing the method of any of the above embodiments.

A computer-readable storage medium of an embodiment of the present invention stores a program for execution by a computer, and executing the program includes performing the method of any of the above embodiments. The computer-readable storage medium may include: read-only memory, random access memory, magnetic or optical disk, and the like.

Additional aspects and advantages of embodiments of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

The above and/or additional aspects and advantages of embodiments of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

fig. 1 is a schematic diagram of a variation curve of the detected Z-axis coordinate of the lens module along the focus tracking direction.

Fig. 2 is a schematic diagram of another variation curve of the detected Z-axis coordinate of the lens module along the focus tracking direction.

Fig. 3 is a perspective view of a carrying device according to an embodiment of the present invention.

Fig. 4 is an exploded view of a carrier according to an embodiment of the present invention.

Fig. 5 is a schematic diagram illustrating a positional relationship between a lens module and a sample according to an embodiment of the present invention.

Fig. 6 is a partial structural schematic diagram of an optical detection system according to an embodiment of the present invention.

Fig. 7 is a schematic flow diagram of an imaging method according to an embodiment of the invention.

Fig. 8 is another schematic flow diagram of an imaging method according to an embodiment of the invention.

Fig. 9 is a further flowchart of the image forming method according to the embodiment of the present invention.

Fig. 10 is an exploded schematic view of a temperature control device according to an embodiment of the present invention.

FIG. 11 is a schematic view of the construction of a water bath chamber and a temperature controlled water bath apparatus according to an embodiment of the present invention.

FIG. 12 is a partially exploded schematic view of a carrier according to an embodiment of the present invention.

Fig. 13 is a schematic structural view of a fluid device according to an embodiment of the present invention.

FIG. 14 is a schematic flow chart of a method for controlling a sequence determination reaction according to an embodiment of the present invention.

Fig. 15 is a block schematic diagram of an image forming apparatus according to an embodiment of the present invention.

Fig. 16 is another block diagram of an image forming apparatus according to an embodiment of the present invention.

FIG. 17 is a block schematic diagram of an optical detection system according to an embodiment of the present invention.

FIG. 18 is a block diagram of a sequence determining system according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, features defined as "first", "second", may explicitly or implicitly include one or more of the described features. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, "connected" is to be understood in a broad sense, e.g., fixedly, detachably or integrally connected; may be mechanically connected, may be electrically connected or may be in communication with each other; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The following disclosure provides many different embodiments or examples for implementing different features of the invention. To simplify the disclosure of the present invention, the components and settings of a specific example are described below. Furthermore, the present invention may repeat reference numerals and/or reference letters in the various examples, which have been repeated for purposes of simplicity and clarity and do not in themselves dictate a relationship between the various embodiments and/or configurations discussed.

In the description of the present invention, it is to be understood that the terms "thickness", "upper", "lower", "front", "rear", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are used only for convenience in 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, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

In the description of the present invention, the term "constant", for example relating to distance, object distance and/or relative position, may be expressed as a change in value, value range or quantity, may be absolutely constant or may be relatively constant, and the term relatively constant is maintained within a certain deviation range or a preset acceptable range. "invariant" with respect to distance, object distance, and/or relative position is relatively invariant, unless otherwise specified. It should be noted that, since the specific data referred to in the description of the present invention are mostly statistically significant, any numerical value expressed in a precise manner represents a range including a range of plus or minus 10% of the numerical value unless otherwise specified, and the description thereof will not be repeated below.

The "sequencing" referred to in the embodiments of the present invention is similar to nucleic acid sequencing, including DNA sequencing and/or RNA sequencing, including long fragment sequencing and/or short fragment sequencing. The so-called "sequencing reaction" is the same as the sequencing reaction.

The embodiment of the invention provides an imaging method, the imaging method is used for an optical detection system, the optical detection system comprises an imaging device 102 and a carrying device 100, the carrying device 100 comprises a temperature control device 301 and a carrying platform 103, the imaging device 102 comprises a lens module 104, the lens module 104 comprises an optical axis OP, the carrying platform 103 is used for carrying a sample 300, and the temperature control device 301 is used for adjusting the temperature of the sample 300. The imaging method comprises the following steps: before image acquisition of the sample 300 by the imaging device 102 or at the time of image acquisition of the sample 300 by the imaging device 102, a range in which temperature fluctuation of the sample 300 is allowed is set by the temperature control device 301 so that a positional fluctuation range of the lens module 104 along the optical axis OP is within a preset range. By using the imaging method, the position fluctuation range of the lens module 104 can be controlled within a preset range, and adverse effects on the imaging device 102 during image acquisition can be reduced or avoided.

Specifically, the temperature control device 301 may be in contact with the sample 300 to set the range of temperature fluctuation of the sample 300.

In some embodiments, the optical detection system is preset with a corresponding relationship between the temperature fluctuation range of the sample 300 and the position fluctuation range of the lens module 104 along the optical axis, and controls the position fluctuation range of the lens module 104 along the optical axis OP to be within a preset range according to the corresponding relationship. Thus, the imaging method can quickly acquire the temperature fluctuation range of the sample 300 corresponding to the position fluctuation range of the lens module 104 along the optical axis being controlled within the preset range.

Specifically, the correspondence relationship between the range of temperature fluctuation of the sample 300 and the range of position fluctuation of the lens module 104 along the optical axis may be stored in the optical detection system. In one example, the correspondence may be referred to in table 1 below.

TABLE 1

It should be noted that the data in table 1 are only used for illustrating the embodiment of the present invention, and should not be construed as limiting the present invention. In other examples, the correspondence between the range of temperature fluctuation of the sample 300 and the range of position fluctuation of the lens module 104 along the optical axis may be specifically set according to actual needs.

In some embodiments, the structure of the carrier 100 is as shown in fig. 3 and 4. The position relationship between the lens module 104 and the sample 300 is shown in fig. 5.

In still other embodiments, referring to fig. 6, the imaging device 102 includes a focusing module 106, and the image acquisition of the sample 300 by the imaging device 102 includes: the sample 300 is focused by the focusing module 106 and the lens module 104. In this way, the imaging device 102 performs focusing during image acquisition, and can acquire a clear sample image.

In some embodiments, referring to fig. 7, focusing includes the steps of: s11, using the focusing module 106 to emit light onto the sample 300 placed on the stage 103; s12, moving the lens module 104 to a first setting position along the optical axis OP; s13, moving the lens module 104 from the first setting position to the sample 300 along the optical axis 300 by a first setting step length and determining whether the focusing module 106 receives the light reflected by the sample 300; when the focusing module 106 receives the light reflected by the sample 300, S14 causes the lens module 104 to move along the optical axis OP by a second set step smaller than the first set step and to perform image acquisition on the sample 300 by using the imaging device 102, and determines whether the sharpness value of the image acquired by the imaging device 102 reaches a set threshold; when the sharpness value of the image reaches the set threshold value, S15 stores the current position of the lens module 104 as the storage position. By utilizing the imaging method, the clear imaging plane of the target object, namely the clear plane/clear plane, can be quickly and accurately found. The method is particularly suitable for devices containing precise optical systems, such as optical detection devices with high power lenses, where clear planes are not easily found.

Specifically, the sample 300 can be understood as a sample in a broad sense or a sample in a narrow sense. For the sample in the broad sense, the sample is a sample to be tested such as each solution, reagent and the like. The sample may be placed on a support device, such as a reaction device (e.g., a chip), a slide, etc., and the support device with the sample is placed on the carrier device 100. For samples in the narrow sense, the sample includes a support device and a sample to be tested on the support device, and the sample can be placed on the carrier device 100 during sequence measurement. Referring to fig. 5, in an embodiment of the focusing step, the sample 300 includes a supporting device 200 and a sample 302 to be measured located on the supporting device 200, the sample 302 to be measured is a biomolecule, such as a nucleic acid, etc., and the lens module 104 is located above the supporting device 200. The support device 200 has a front panel 202 and a rear panel (lower panel), each having two surfaces, with a sample 302 to be tested attached to the upper surface of the lower panel, i.e., the sample 302 to be tested is located below the lower surface 204 of the front panel 202. In the embodiment of the invention, since the imaging device 102 is used to acquire the image of the sample 302 to be measured, and the sample 302 to be measured is located below the lower surface 204 of the front panel 202 of the supporting device 200, when the focusing process starts, the lens module 104 moves to find the medium interface 204 where the sample 302 to be measured is located, so as to improve the success rate of acquiring a clear image by the imaging device 102. In the embodiment of the present invention, the sample 302 to be measured is a solution, the front panel 202 of the supporting device 200 is glass, and the medium interface 204 between the supporting device 200 and the sample 302 to be measured is the lower surface 204 of the front panel 202 of the supporting device 200, i.e. the interface between the glass and the liquid. The sample 302 to be tested whose image needs to be acquired by the imaging device 102 is located below the lower surface 204 of the front panel 202, and the clear surface for clearly imaging the sample 302 to be tested is determined and found according to the image acquired by the imaging device 102, which may be referred to as focusing. In one example, the front panel 202 of the sample 302 to be tested has a thickness of 0.175 mm.

In some embodiments, the support device 200 can be a slide on which the sample 302 to be tested is placed, or the sample 302 to be tested is clamped between two slides. In some embodiments, the support device 200 may be a reaction device, such as a chip with a sandwich structure having a top and a bottom carrying panels, on which the sample 302 to be tested is disposed.

In some embodiments, referring to fig. 6, the imaging device 102 includes a microscope 107 and a camera 108, the lens module 104 includes an objective lens 110 of the microscope and a lens module 112 of the camera 108, the focusing module 106 can be fixed with the lens module 112 of the camera 108 by a dichroic beam splitter 114(dichroic beam splitter), and the dichroic beam splitter 114 is located between the lens module 112 of the camera 108 and the objective lens 110. The dichroic beam splitter 114 includes a dual C-shaped beam splitter (dual C-mount splitter). The dichroic beam splitter 114 reflects the light emitted from the focusing module 106 to the objective lens 110 and allows visible light to pass through and enter the camera 108 through the lens module 112 of the camera 108, as shown in fig. 6.

In the embodiment of the invention, the movement of the lens module 104 may refer to the movement of the objective lens 110, and the position of the lens module 104 may refer to the position of the objective lens 110. In other embodiments, other lenses of the lens module 104 may be selectively moved to achieve focus. In addition, the microscope 107 further includes a tube lens 111(tube lens) between the objective lens 110 and the camera 108.

In some embodiments, the stage 103 can move the sample 200 in a plane (e.g., XY plane) perpendicular to the optical axis OP (e.g., Z axis) of the lens module 104, and/or can move the sample 300 along the optical axis OP (e.g., Z axis) of the lens module 104.

In some embodiments, the plane in which the stage 103 drives the sample 300 to move is not perpendicular to the optical axis OP, i.e. the included angle between the moving plane of the sample and the XY plane is not 0, and the imaging method is still suitable.

In addition, the imaging device 102 can also drive the objective lens 110 to move along the optical axis OP of the lens module 104 for focusing. In some examples, the imaging device 102 drives the objective lens 110 to move using an actuator such as a stepper motor or a voice coil motor.

In some embodiments, when establishing the coordinate system, as shown in fig. 5, the positions of the objective lens 110, the stage 103, and the sample 300 may be set on the negative axis of the Z-axis, and the first set position may be a coordinate position on the negative axis of the Z-axis. It is understood that, in other embodiments, the relationship between the coordinate system and the camera and the objective lens 110 may be adjusted according to actual situations, and is not limited in particular.

In one example, the imaging device 102 comprises a total internal reflection fluorescence microscope, the objective lens 110 is at 60 times magnification, and the first set step size S1 is 0.01 mm. Thus, the first setting step S1 is more suitable, since S1 is too large to cross the acceptable focusing range, and S1 is too small to increase the time overhead.

When the focusing module 106 does not receive the light reflected by the sample 300, the lens module 104 is moved to the sample 300 along the optical axis OP by a first set step.

In some embodiments, when the sharpness value of the image does not reach the set threshold, the lens module 104 is moved along the optical axis OP by a second set step.

In certain embodiments, the optical detection system may be applied to, or comprise, a sequencing system.

In some embodiments, when the lens module 104 moves, it is determined whether the current position of the lens module 104 exceeds a second predetermined position; when the current position of the lens module 104 exceeds the second setting position, the lens module 104 is stopped to move or a focusing step is performed. Thus, the first setting position and the second setting position can limit the moving range of the lens module 104, so that the lens module 104 can stop moving when focusing is failed, thereby avoiding waste of resources or damage of equipment, or refocusing the lens module 104 when focusing is failed, and improving the automation of the imaging method.

In some embodiments, such as in a total internal reflection imaging system, the arrangement is adjusted to minimize the range of motion of the lens module 104 in order to achieve a fast media interface. For example, in the total internal reflection imaging device with a 60-fold objective lens, the moving range of the lens module 104 can be set to 200 μm ± 10 μm or [190 μm, 250 μm ] according to the optical path characteristics and empirical summary.

In some embodiments, depending on the determined range of movement and the setting of either the second set position or the first set position, another set position may be determined. In one example, the second setting position is set to reflect the lowest position of the upper surface 205 of the front panel 202 of the apparatus 200 and then the next depth of field, and the moving range of the lens module 104 is set to 250 μm, so that the first setting position is determined. In the present example, the coordinate position corresponding to the position of the next depth of field size is a position that becomes smaller in the negative Z-axis direction.

Specifically, in the embodiment of the present invention, the movement range is one section on the negative axis of the Z axis. In one example, the first set position is nearlimit, the second set position is farlimit, and the coordinate positions corresponding to nearlimit and farlimit are both located on the negative axis of the Z-axis, where nearlimit is-6000 um, and farlimit is-6350 um. The size of the range of motion defined between nearlimit and farlimit is 350 um. Therefore, when the coordinate position corresponding to the current position of the lens module 104 is smaller than the coordinate position corresponding to the second set position, it is determined that the current position of the lens module 104 exceeds the second set position. In fig. 5, the position of farlimit is the position of the depth of field L next to the lowest position of the upper surface 205 of the front panel 202 of the reaction apparatus 200. The depth of field L is the depth of field of the lens module 104.

It should be noted that, in other embodiments, the coordinate position corresponding to the first setting position and/or the second setting position may be specifically set according to the actual situation, and is not specifically limited herein.

In some embodiments, the focusing module 106 includes a first light source 116 and a light sensor 118, the first light source 116 is configured to emit light onto the sample 300, and the light sensor 118 is configured to receive light reflected by the sample 300. Thus, the light emitting and receiving of the focusing module 106 can be realized.

Specifically, in the embodiment of the present invention, the first light source 116 may be an infrared light source 116, and the light sensor 118 may be a photo diode (photo diode), so that the cost is low and the accuracy of the detection is high. Infrared light emitted by the first light source 116 enters the objective lens 110 via reflection by the dichroic beam splitter and is projected onto the sample 300 via the objective lens 110. The sample 300 may reflect the infrared light projected through the objective lens 110. In an embodiment of the focusing step, when the sample 300 comprises the supporting device 200 and the sample 302 to be measured, the received light reflected by the sample 300 is the light reflected by the lower surface 204 of the front panel of the supporting device 200.

Whether the infrared light reflected by the sample 300 can enter the objective lens 110 and be received by the light sensor 118 depends primarily on the distance between the objective lens 110 and the sample 300. Therefore, when the focusing module 106 receives the infrared light reflected by the sample 300, it can be determined that the distance between the objective lens 110 and the sample 300 is within the suitable range for optical imaging, and the distance can be used for imaging of the imaging device 102. In one example, the distance is 20-40 um.

At this time, the lens module 104 is moved by a second setting step smaller than the first setting step, so that the optical detection system can search the optimal imaging position of the lens module 104 in a smaller range.

In some embodiments, the sharpness value of the image may be used as an evaluation value (evaluation value) for focusing the image. In one embodiment, determining whether the sharpness value of the image captured by the imaging device 102 reaches a set threshold may be performed by a hill-climbing algorithm of image processing. Whether the sharpness value reaches the maximum value at the peak of the sharpness value is judged by calculating the sharpness value of the image output by the imaging device 102 when the objective lens 110 is at each position, and whether the lens module 104 reaches the position of the clear plane when the imaging device 102 images is further judged. It will be appreciated that in other embodiments, other image processing algorithms may be used to determine whether the sharpness value has reached a maximum at the peak.

When the sharpness value of the image reaches the set threshold value, the current position of the lens module 104 is saved as the saving position, so that the imaging device 102 can output a clear image when the sequence measurement reaction is performed to collect the image.

In some embodiments, referring to fig. 8, when the focusing module 106 receives the light reflected by the sample 300, the focusing further comprises: s16, moving the lens module 104 along the optical axis OP toward the sample 300 by a third setting step that is smaller than the first setting step and larger than the second setting step, calculating a first light intensity parameter according to the light intensity of the light received by the focusing module 106, and determining whether the first light intensity parameter is larger than a first setting light intensity threshold; when the first light intensity parameter is greater than the first set light intensity threshold, step S14 is performed. In this way, by comparing the first light intensity parameter with the first set light intensity threshold, the interference of the light signal with very weak contrast with the reflected light of the medium interface to focusing can be eliminated.

When the first light intensity parameter is not greater than the first set light intensity threshold, the lens module 104 is moved to the sample 300 along the optical axis OP by a third set step length.

In some embodiments, the focusing module 106 includes two light sensors 118, the two light sensors 118 are configured to receive light reflected by the sample 300, and the first light intensity parameter is an average of light intensities of the light received by the two light sensors 118. In this way, the first light intensity parameter is calculated by the average of the light intensities of the light received by the two light sensors 118, so that it is more accurate to exclude weak light signals.

Specifically, the first light intensity parameter may be set to SUM, i.e., SUM ═ PD1+ PD2)/2, and PD1 and PD2 respectively indicate the light intensities of the light received by the two light sensors 118. In one example, the first set light intensity threshold nSum is 40.

In one example, the third set step size S2 is 0.005 mm. It is understood that, in other examples, the third setting step may also take other values, and is not limited in particular.

In some embodiments, referring to fig. 9, when the focusing module 106 receives the light reflected by the sample 300, the focusing further comprises the following steps: s16, moving the lens module 104 along the optical axis OP toward the sample 300 by a third setting step that is smaller than the first setting step and larger than the second setting step, calculating a first light intensity parameter according to the light intensity of the light received by the focusing module 106, and determining whether the first light intensity parameter is larger than a first setting light intensity threshold; when the first light intensity parameter is greater than the first set light intensity threshold, S17, the lens module 104 moves along the optical axis OP toward the sample 300 by a fourth set step that is smaller than the third set step and larger than the second set step, and calculates a second light intensity parameter according to the light intensity of the light received by the focusing module 106, and determines whether the second light intensity parameter is smaller than the second set light intensity threshold; when the second light intensity parameter is smaller than the second set light intensity threshold, step S14 is performed. Therefore, by comparing the first light intensity parameter with the first set light intensity threshold value, the interference of light signals with very weak contrast with the reflected light of the medium interface on focusing/focusing can be eliminated; and the strong reflected light signals at the position of the non-medium interface, such as the interference of the light signals reflected by the oil surface/air of the objective lens 110 on focusing/focusing can be eliminated by comparing the second light intensity parameter with the second set light intensity threshold.

When the first light intensity parameter is not greater than the first set light intensity threshold, the lens module 104 is moved to the sample 300 along the optical axis OP by a third set step length. When the second light intensity parameter is not less than the second set light intensity threshold, the lens module 104 is moved to the sample 300 along the optical axis OP by a fourth set step length.

In one example, the third setting step S2 is 0.005mm, and the fourth setting step S3 is 0.002 mm. It is understood that, in other examples, the third setting step and the fourth setting step may also adopt other values, and are not limited in particular.

In some embodiments, the focusing module 106 includes two light sensors 118, the two light sensors 118 are configured to receive light reflected by the sample 300, the first light intensity parameter is an average value of light intensities of the light received by the two light sensors 118, the light intensities of the light received by the two light sensors 118 have a first difference, and the second light intensity parameter is a difference between the first difference and the set compensation value. In this manner, the second light intensity parameter is calculated from the light intensities of the light received by the two light sensors 118, so that the light signal excluding the strong reflection is more accurate.

Specifically, the first light intensity parameter may be set to SUM, i.e., SUM ═ PD1+ PD2)/2, and PD1 and PD2 respectively indicate the light intensities of the light received by the two light sensors 118. In one example, the first set light intensity threshold nSum is 40. The difference may be set to err and the offset may be set to err ═ offset (PD1-PD2) -offset. In an ideal situation, the first difference value may be zero. In one example, the second set light intensity threshold value nrerr is 10 and the offset is 30.

In some embodiments, when the lens module 104 is moved by the second set step length, it is determined whether a first sharpness value of a pattern corresponding to a current position of the lens module 104 is greater than a second sharpness value of an image corresponding to a previous position of the lens module 104; when the first sharpness value is greater than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is greater than the set difference, the lens module 104 is made to continue moving towards the sample 300 along the optical axis OP by a second set step length; when the first sharpness value is greater than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is smaller than the set difference, the lens module 104 continues to move along the optical axis OP toward the sample 300 by a fifth set step smaller than the second set step so that the sharpness value of the image acquired by the imaging device 102 reaches the set threshold; when the second sharpness value is greater than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is greater than the set difference, the lens module 104 is moved away from the sample 300 along the optical axis OP by a second set step length; when the second sharpness value is greater than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is smaller than the set difference, the lens module 104 is moved away from the sample 300 along the optical axis OP by a fifth set step length so that the sharpness value of the image acquired by the imaging device 102 reaches the set threshold. Therefore, the position of the lens module 104 corresponding to the peak of the sharpness value can be accurately found, so that the image output by the imaging device is clear.

Specifically, the second set step may be taken as a coarse step Z1, the fifth set step may be taken as a fine step Z2, and a coarse adjustment range Z3 may be set. The coarse adjustment range Z3 is set to stop the movement of the lens module 104 when the sharpness value of the image fails to reach the set threshold, thereby saving resources.

Taking the current position of the lens module 104 as the starting point T, the coarse adjustment range Z3 is the adjustment range, i.e. the adjustment range on the Z axis is (T, T + Z3). The lens module 104 is moved in a first direction (e.g., a direction approaching the sample 300 along the optical axis OP) by a step Z1 within a range of (T, T + Z3), and a first sharpness value R1 of an image captured by the imaging device 102 at a current position of the lens module 104 is compared with a second sharpness value R2 of an image captured by the imaging device 102 at a previous position of the lens module 104.

When R1> R2 and R1-R2> R0, which illustrate the sharpness value of the image being closer to the set threshold and farther from the set threshold, the lens module 104 continues to move in the first direction by the step Z1 to quickly approach the set threshold.

When R1> R2 and R1-R2< R0, which indicate that the sharpness value of the image is close to the set threshold and closer to the set threshold, the lens module 104 is moved in the first direction by a step Z2 and is moved closer to the set threshold by a smaller step.

When R2> R1 and R2-R1> R0, which indicate that the sharpness value of the image has crossed the set threshold and is farther from the set threshold, the lens module 104 is moved in a second direction opposite to the first direction (e.g., a direction away from the sample 300 along the optical axis OP) by a step Z1 to quickly approach the set threshold.

When R2> R1 and R2-R1< R0, which indicates that the sharpness value of the image has crossed the set threshold and is closer to the set threshold, the lens module 104 is moved in a second direction opposite to the first direction by a step Z2, and is closer to the set threshold by a smaller step.

In some embodiments, the fifth setting step size can be adjusted to adapt to the step size approaching the setting threshold value, which is not too large or too small.

In one example, T is 0, Z1 is 100, Z2 is 40, Z3 is 2100, and the adjustment range is (0,2100). It should be noted that the above values are measurement values used when the lens module 104 is moved during the image capturing process performed by the imaging device 102, and the measurement values are light intensity-related.

In certain embodiments, image acquisition of the sample 300 using the imaging device 102 comprises: the lens module 104 is used to focus the sample 300. In this manner, it is ensured that the images acquired by the imaging device 102 at different positions of the sample 300 remain clear.

In some embodiments, the step of tracking comprises the steps of: when the lens module 104 is in the storage position, acquiring the relative position of the lens module 104 and the sample 300; when the stage 103 moves the sample 300, the lens module 104 is controlled to keep the relative position unchanged. In this way, when the imaging device 102 collects images at different positions of the sample 300, the collected images are kept clear, and tracking is realized.

Specifically, the sample 300 may be tilted due to physical errors of the stage 103 and/or the sample 300, and thus, when the stage 103 moves the sample 300, the distance between the lens module 104 and the different positions of the surface of the sample 300 may slightly change. Therefore, when the sample 300 moves relative to the optical axis OP of the lens module 104, the imaging position of the imaging device 102 on the sample 300 is always kept at the clear plane position. This process is called focus tracking.

The stage 103 is used to move the sample 300, including the sample 300 along an axis X1 parallel to the X-axis, and the sample 300 along an axis Y1 parallel to the Y-axis, and the sample 300 along a plane X1Y1 defined by the X1 axis and the Y1 axis, and the sample 300 along a plane XY defined by the X-axis and the Y-axis, and the sample 300 along a plane oblique to the X-axis and the Y-axis.

In some embodiments, when the stage 103 is used to drive the sample 300 to move, it is determined whether the current position of the lens module 104 exceeds a third set position; when the current position of the lens module 104 exceeds the third setting position, the stage 103 is used to drive the sample 300 to move along the optical axis OP and a focusing step is performed; when the moving times reach the set times and the current position of the lens module 104 still exceeds the third set position, it is determined that the focus tracking fails. Thus, the limitation of the third setting position and the moving times can make the lens module 104 refocus when the focus tracking fails.

Specifically, in the present example, the third setting position may be nPos, a coordinate position corresponding to the nPos is on a negative axis of the Z-axis, and the coordinate position corresponding to the nPos is greater than a coordinate position corresponding to the second setting position farlimiit. When the coordinate position corresponding to the current position of the lens module 104 is smaller than the coordinate position corresponding to the third setting position, it is determined that the current position of the lens module 104 exceeds the third setting position.

When it is determined that the current position of the lens module 104 exceeds the third predetermined position for the first time, refocusing is performed to adjust the position of the lens module 104 to try to successfully follow up. In the process of focusing, if the number of times of moving the lens module 104 reaches the set number of times, the current position of the lens module 104 still exceeds the third set position, and then the lens module cannot focus, and it is determined that the focusing fails, and the focus is paused and refocused to find the clear plane.

The coordinate position corresponding to the third setting position is an empirical value, and when the value is smaller than the empirical value, the image acquired by the imaging device 102 is blurred and fails to be focused. The setting times are empirical values and can be specifically set according to actual conditions.

In some embodiments, when the current position of the lens module 104 does not exceed the third set position, the relative position is determined to be unchanged. In some embodiments, the relative position includes a relative distance and a relative direction. Further, for simplicity, the relative position may refer to a relative distance, and the invariant relative position refers to the invariant object distance of the imaging system of the imaging device 102, which may enable different positions of the sample 300 to be clearly imaged by the imaging device.

In some embodiments, referring to fig. 3-4 and 10-12, the carrier 100 includes a base plate 101 and a resilient support assembly 40. The carrier 103 is fixed on the bottom plate 101 and has a receiving groove 242, and a through hole 2422 is formed at the bottom of the receiving groove 242. The temperature control device 301 is connected to the sample 300 in the container 242 through the through hole 2422 and is elastically supported on the base plate 101 by the elastic support member 40.

In the carrying device 100 according to the embodiment of the invention, the temperature control device 301 is elastically supported on the bottom plate 101 by the elastic supporting component 40, so that when the sample 300 is loaded into the accommodating groove 242, the contact between the sample 300 and the temperature control device 301 and the bottom plate 101 is elastic contact, thereby effectively preventing the sample 300 from being damaged during assembly.

Specifically, when stage 103 does not yet have device sample 300, elastic support assembly 40 pushes temperature control device 301 toward container 242, so that temperature control device 301 protrudes relative to the bottom surface of container 242. When the sample 300 is loaded in the containing groove 242, the sample 300 is pressed toward the temperature control device 301 by the clamping frame 221 of the stage 103, and at the same time, the pressed temperature control device 301 compresses the elastic supporting member 40 downward, so that the elastic supporting member 40 generates an elastic force, and finally the sample 300 is locked on the stage 103. When the sample 300 is loaded and the sample 300 is locked, the sample 300 and the temperature control device 301 are in elastic contact with the bottom plate 101, thereby effectively preventing the sample 300 from being damaged during the assembling and sequencing processes.

In some embodiments, the elastic supporting assembly 40 includes a guiding cylinder 43 and an elastic member 44, the temperature control device 301 includes a temperature control portion 312 and a guiding column 341, the guiding column 341 is disposed on a side of the temperature control portion 312 away from the containing groove 242, the guiding cylinder 43 is fixed on the bottom plate 101, the guiding column 341 penetrates through the elastic member 44 and the guiding cylinder 43, and the elastic member 44 elastically abuts between the temperature control portion 312 and the guiding cylinder 43. Thus, the elastic member 44 can provide an elastic force, and the cooperation between the guiding cylinder 43 and the guiding column 341 can ensure that the movement of the temperature control device 301 is stable.

It can be understood that when the sample 300 is loaded into the container 242, it contacts the temperature control device 301 and is pressed down, the elastic member 44 is compressed, the reaction force of the compressed elastic member 44 provides sufficient contact between the sample 300 and the temperature control device 301, and at the same time, the elastic contact provided by the elastic member 44 ensures that the sample 300 is not easily damaged during the pressing down process.

In some embodiments, the elastic member 44 may be a spring, so that the manufacturing cost of the carrier device 100 can be reduced.

In some embodiments, the elastic member 44 may be a rubber cylinder or other elastic element, which is not limited herein.

In some embodiments, the guide cylinder 43 is a linear bearing, and the guide post 341 is in sliding contact with a ball of the linear bearing. Thus, the linear bearing can reduce the resistance when the temperature control device 301 moves while guiding the movement of the temperature control device 301.

It can be understood that during the pressing down of the sample 300, the temperature control device 301 is pressed to move downward, that is, the guiding column 341 needs to slide downward, and the ball of the linear bearing can reduce friction when the guiding column 341 slides downward.

Referring to fig. 4 and 10, in some embodiments, the temperature control device 301 includes a fixing plate 321, a temperature conducting plate 322, a temperature control element 323, and a guiding column 341, the temperature control element 323 is sandwiched between the fixing plate 321 and the temperature conducting plate 322, the temperature control element 323 is in contact with the temperature conducting plate 322 and the fixing plate 321, the temperature conducting plate 322 is used for contacting the sample 300 loaded in the containing cavity 242, the guiding column 341 is disposed on a surface of the fixing plate 321 away from the temperature control element 323, and the guiding column 341 penetrates through the elastic supporting member 40. Thus, the function of the temperature control device 301 is realized by using a simpler structure, and the cost of the carrying device 100 is reduced.

It will be appreciated that the fixed plate 321 provides support for the temperature control element 323 and that the temperature conduction plate 322 transfers the temperature generated by the temperature control element 323 to the sample 300, thereby achieving temperature control of the sample 300. The guiding column 341 penetrates the elastic supporting member 40, and the sample 300 and the temperature control device 301 are brought into close contact by the elastic force of the elastic supporting member 40.

In some embodiments, the temperature conducting plate 322 and the fixing plate 321 may be made of a metal material for conducting the temperature of the temperature control element 323.

In one example, the temperature control element 323 is a peltier. The temperature control element 323 can realize cooling and heating by using the peltier effect. For example, by applying currents in different directions to the temperature control element 323, the upper surface of the temperature control element 323 is cooled and the lower surface is heated, or the upper surface of the temperature control element 323 is heated and the lower surface is cooled. The temperature conduction plate 322 in contact with the upper surface of the temperature control element 323 conducts the temperature of the upper surface of the temperature control element 323 to the sample 300, thereby achieving temperature control of the sample 300.

In some embodiments, a heat conducting layer may be provided between the temperature conducting plate 322 and the temperature control element 323, the heat conducting layer conducting the temperature of the temperature control element 323 to the temperature conducting plate 322. In this way, the thermal conductivity between the temperature control element 323 and the temperature transmission plate 322 is improved. In one example, the thermally conductive layer is a silicone layer.

In some embodiments, the temperature control device 301 further comprises a water bath chamber 324 disposed on a surface of the fixing plate 321 remote from the temperature control element 323, the water bath chamber 324 being spaced apart from the guide posts 341. Thus, when the temperature control element 323 operates, the water bath chamber 324 can take away heat of the temperature control element 323 through the fixing plate 321 in time.

In some embodiments, the temperature control device 301 includes a temperature sensor 325 disposed on the temperature conductive plate 322. Thus, the temperature of the temperature conduction plate 322 can be detected, and accurate temperature control of the sample 300 is facilitated.

Specifically, when the carrier 100 is in operation, since the temperature conduction plate 322 is in close contact with the sample 300, and the temperature of the temperature conduction plate 322 is equivalent to the temperature of the sample 300, the temperature sensor 325 can obtain the temperature of the sample 300 and feed back the temperature to the external control device, so that the external control device can control the temperature of the sample 300 according to the feedback of the temperature sensor 325.

In the embodiment of the present invention, the temperature conduction plate 322 may be formed with a receiving hole, and the temperature sensor 325 is inserted into the receiving hole, so that the temperature of the sample 300 can be monitored more accurately. In one embodiment, the carrier 100 is capable of achieving a sample 300 surface temperature accuracy of ± 0.1 ℃ or ± 0.5 ℃; the surface temperature fluctuation degree of the sample 300 is not more than 0.1 ℃ or 0.5 ℃; the temperature rising time from the room temperature of 25 ℃ to 65 ℃ is not more than 1min, and the temperature falling time from the temperature of 65 ℃ to the room temperature of 25 ℃ is not more than 1.5 min. The sample 300 is accurate in temperature control, the biochemical reaction efficiency can be improved, and the testing time is shortened.

Referring to fig. 11, in some embodiments, bath 324 includes heat sink 3242, cover 3244, inlet 3246, and outlet 3248. The heat radiating plate 3242 is provided with a flow channel, and the heat radiating plate 3242 contacts the fixed plate 321. Cover plate 3244 is connected with heat dissipation plate 3242 and covers the channel slot to form a liquid cavity, the liquid cavity is used for containing cooling liquid, and a liquid inlet and a liquid outlet which are communicated with the liquid cavity are formed in cover plate 3244. The liquid inlet connector 3246 is connected with a liquid inlet. The liquid outlet connector 3248 is connected with a liquid outlet. Thus, an efficient heat dissipation structure of the water bath chamber 324 is realized.

It will be appreciated that the water bath chamber 324 is used to provide a coolant circulation for dissipating heat from the lower surface of the temperature control element 323 which is in contact with the fixed plate 321. The channel groove provided in the heat radiating plate 3242 can increase the contact area between the heat radiating plate 3242 and the fixing plate 321, thereby improving heat radiating efficiency.

In certain embodiments, the cooling fluid may be water. Thus, the cost of the carrier device 100 can be reduced.

In some embodiments, the cooling fluid may be a specially made cooling fluid, without limitation. The specially made cooling liquid can ensure that the heat conduction capability reaches a more ideal state.

Referring again to fig. 11, in some embodiments, temperature control device 301 includes a temperature controlled water bath apparatus 326, and temperature controlled water bath apparatus 326 includes a heat sink 3262, an infusion pump 3264, and a cooling device 3266. The heat sink 3262 has a flow path through which a cooling liquid flows, and an inlet of the flow path is connected to the liquid outlet connector 3248. An infusion pump 3264 connects the outlet of the flow path to an inlet connector 3246. The cooling device 3266 is used to cool the heat sink 3262. In this manner, heat from the water bath chamber 324 can be carried to the temperature controlled water bath 326 and dissipated through the heat sink 3262.

In particular, the temperature-controlled water bath apparatus 326 is used to accelerate the heat exchange between the cooling fluid and the external environment, thereby ensuring a rapid cooling of the cooling fluid. The cooling fins 3262 are used to exchange heat between the cooling fluid and the external environment, the liquid delivery pump 3264 is used to facilitate circulation of the cooling fluid, and the cooling device 3266 is used to accelerate the heat exchange of the cooling fluid in the cooling fins 3262.

In certain embodiments, the cooling device 3266 can be a fan. The heat exchange of the coolant in the heat radiating fins 3262 is accelerated by blowing air to the heat radiating fins 3262 by a fan to increase the convection of the air.

In certain embodiments, temperature control device 301 includes thermal insulation 327. The heat insulator 327 is disposed between the temperature conductive plate 322 and the fixing plate 321. Thus, it is able to avoid the temperature conduction plate 322 from being interfered by the temperature of the fixing plate 321 to cause inaccurate temperature control of the sample 300.

Specifically, the temperature transmission plate 322 and the fixing plate 321 are respectively in contact with two different surfaces of the temperature control element 323, and the temperatures of the two surfaces are different during the operation of the temperature control element 323. In order to ensure that the temperature conduction plate 322 accurately conducts the temperature of the temperature control element 323 to the sample 300, the heat insulator 327 is used to block the temperature transmission between the temperature conduction plate 322 and the fixing plate 321, thereby ensuring that the temperature of the temperature conduction plate 322 is not affected by the temperature of the fixing plate 321.

In some embodiments, the insulation 327 may be insulation cotton. The thermal insulation cotton can realize thermal insulation and simultaneously avoid damaging the temperature control element 323 between the temperature conduction plate 322 and the fixed plate 321.

In some embodiments, temperature control device 301 comprises a surface 328 for contacting sample 300, surface 328 being a matte black surface. Thus, the laser reflection generated by the carrier 100 during operation can be prevented from adversely affecting the imaging of the sample 300.

Specifically, the flow channel in the sample 300 is transparent, and when the carrying device 100 is operated, laser light is emitted to the sample 300 to excite the sample in the sample 300 to emit fluorescence, and an image of the sample is formed by collecting the fluorescence. By providing the surface 328 in contact with the sample 300 as a matte black surface, the reflection of the laser light is effectively reduced.

In some embodiments, surface 328 is the upper surface of temperature-conducting plate 322 that is in contact with sample 300.

In some embodiments, the carrier 103 includes a clamping frame 221 and a supporting base 241, the clamping frame 221 is rotatably connected to the supporting base 241, the supporting base 241 has an accommodating groove 242, and the supporting base 241 is disposed on the bottom plate 101. Thus, the loading and unloading of the sample 300 can be facilitated by the rotation of the clamping frame 221 relative to the supporting base 241.

Specifically, when the sample 300 is placed in the container 242, the clamping frame 221 may be rotated to press the sample 300 toward the temperature control device 301 by the rotational connection of the clamping frame 221 and the support base 241, thereby ensuring sufficient contact between the sample 300 and the temperature control device 301. Then, the clamping frame 221 is locked to the supporting base 241 by, for example, a snap-fit manner, so as to compress the sample 300, thereby ensuring the stability of the sample 300 during the sequencing process. A torsion spring may be disposed at a connection point between the clamping frame 221 and the supporting seat 241, and when the clamping frame 221 is unlocked, the torsion spring may drive the clamping frame 221 to open relative to the supporting seat 241.

Referring to fig. 12, in some embodiments, a button 246 is disposed on the supporting base 241, and the button 246 is connected to a buckle 248. In this way, the button 246 can control the movement state of the catch 248, and the clamping frame 221 can be unlocked by releasing the locked state of the clamping frame 221 and the support base 241.

Specifically, when the button 246 is pressed, the button 246 may lever the clip 248 away from the clamping frame 221, so as to unlock the clamping frame 221 and the clip 248, and thus the assembly or disassembly of the sample 300 may be achieved. When button 246 is released, catch 248 loses force and catch 248 resets. When the clamping frame 221 is closed, the clamping frame 221 is clamped with the buckle 248 again to lock the clamping frame 221.

Referring to fig. 12 again, in some embodiments, the supporting base 241 includes a panel 244, the panel 244 is provided with a button through hole 2442, the button 246 is disposed through the button through hole 2442, the bottom of the button 246 is protruded with a flange 2462, and the flange 2462 abuts against the lower surface of the panel 244.

In this manner, the panel 244 can be pressed against the flange 2462, thereby allowing smooth depression and repositioning of the button 246, and allowing the button 246 to be repositioned with limited resistance to escape.

In some embodiments, referring to fig. 13, fig. 3, fig. 4 and fig. 6, a method for controlling a sequence determination reaction is provided, in which a sequence determination system is used to control the sequence determination reaction, the sequence determination system includes an optical detection system, the optical detection system includes an imaging device 102 and a carrying device 100, the carrying device 100 includes a temperature control device 301 and a stage 103, the imaging device 102 includes a lens module 104, the lens module 104 includes an optical axis OP, the stage 103 is used to carry a sample, and the temperature control device 301 is used to adjust a temperature of the sample. The sequencing reaction includes image acquisition of the sample using the imaging device 102. The method for controlling the sequencing reaction comprises the following steps: before the sequence measurement reaction is performed by the sequence measurement system or when the sequence measurement is performed by the sequence measurement system, the temperature control device 301 sets a range in which the temperature fluctuation of the sample is allowed so that the position fluctuation range of the lens module 104 along the optical axis OP is within a preset range. By using the method for determining the reaction by the control sequence, the position fluctuation range of the lens module 104 can be controlled within a preset range, and adverse effects on the imaging device 102 during image acquisition can be reduced or avoided.

In some embodiments, the optical detection system is preset with a corresponding relationship between the temperature fluctuation range of the sample 300 and the position fluctuation range of the lens module 104 along the optical axis, and controls the position fluctuation range of the lens module 104 along the optical axis OP to be within a preset range according to the corresponding relationship. Thus, the method for controlling the sequence determination reaction can quickly acquire the temperature fluctuation range of the sample 300 corresponding to the position fluctuation range of the lens module 104 along the optical axis being controlled within the preset range.

Specifically, the correspondence relationship between the range of temperature fluctuation of the sample 300 and the range of position fluctuation of the lens module 104 along the optical axis may be stored in the optical detection system. In one example, the correspondence referred to may be found in table 1. In some embodiments, the imaging device 102 includes a focusing module 106, and the image acquisition of the sample 300 by the imaging device 102 includes: the sample 300 is focused by the focusing module 106 and the lens module 104.

It should be noted that the above explanation of the focusing embodiment and the advantageous effects in the optical detection system is also applicable to the focusing in the sequencer system of the present embodiment, and is not detailed here to avoid redundancy.

In some embodiments, referring to fig. 14, the sequence determination reaction includes a first biochemical reaction and a second biochemical reaction, the first biochemical reaction and the second biochemical reaction are performed on the reaction device 200, the sequence determination system includes a fluidic device 500, the fluidic device 500 is connected to the reaction device 200, the reaction device 200 includes a first unit 41 and a second unit 42, the sample 300 is placed on the first unit 41 and the second unit 42, a repeated execution unit S112 included in the sequence determination reaction is defined as a second biochemical reaction-a first biochemical reaction-image collection, and the method for controlling the sequence determination reaction includes, after the following initial step S111 is completed, allowing the imaging device 102 to collect an image of the sample 300 of another unit while one of the first unit 41 and the second unit 42 is subjected to the second biochemical reaction and the first biochemical reaction of the sample 300 by the fluidic device 500, the initial step S111 includes the steps of: a subjecting the sample 300 in one of the first unit 41 and the second unit 42 to a first biochemical reaction using the fluidic device 500, b performing image acquisition of the sample 300 in the unit after performing the first biochemical reaction using the imaging device 102, and c subjecting the sample 300 in the other of the first unit 41 and the second unit 42 to the first biochemical reaction using the fluidic device 500. The method for controlling the sequence determination reaction is based on the sequence determination reaction, the reaction device 200 is divided into at least two units, one unit performs biochemical reaction by using the fluid device 500, and the other unit performs image acquisition, namely image acquisition by using the imaging device 102, so that the time for sequence determination can be reduced, and the sequence determination efficiency is improved.

Specifically, in the embodiment of the method of controlling a sequence determination reaction shown in fig. 14, the sample 300 is a sample to be measured, and the sample 300 is placed on the reaction device 200 (support device). In an embodiment of the present invention, the reaction device 200 with the sample 300 may also be placed on the carrier device 100 to perform a sequencing reaction.

An explanation of the embodiment and advantageous effects of the carrier 100 in the method of controlling a sequencing reaction may be referred to the embodiment of the carrier 100 in the optical detection system described above, and will not be described in detail here to avoid redundancy.

The inventor divides the reaction device into at least two units based on the time difference of biochemical reaction and information collection in the sequencing reaction and the number of imaging devices in the reaction device and the sequencing system, and makes the method for carrying out the sequencing reaction by all or part of the parallel control calling device/system which can be executed by the computer, thereby fully utilizing the time difference of main steps in the sequencing reaction and greatly improving the sequencing reaction efficiency.

Generally, the device/system required for conducting the sequencing reaction, in terms of hardware cost, the cost of the imaging device/system is greater than that of the fluidics device/system, which is greater than that of the reaction device/chip. By using the method of the invention to control the sequence determination reaction, the imaging device/system, the fluid device/system and the reaction device can be fully utilized, and the sequencing cost is further reduced.

Specifically, in some embodiments, the reaction apparatus 200 may be a chip, and the first unit 41 and the second unit 42 of the reaction apparatus 200 each include a plurality of channels (channels), and after the initial step S111, the channels of the first unit 41 and the channels of the second unit 42 are staggered, asynchronous, and do not affect each other in the sequencing reaction. For example, when a biochemical reaction is required on a sample in the first unit 41, the fluidic device 500 will deliver a reagent for the reaction to the first unit 41 without the same reagent entering the second unit 42, and vice versa.

In one example, nucleic acid sequencing is performed on a single molecule sequencing platform using Total Internal Reflection (TIRF) optics to estimate the number of image acquisitions required for the amount of raw data to be approximately 300 fields of view (FOV) based on empirical values of the amount of data required for subsequent genetic information analysis and the proportion of valid data after processing. In one round of sequencing reaction, the time required for controlling the movement of the reaction device 200 and the acquisition of 300 FOVs by the imaging device 102 is approximately equal to the sum of the times of the first biochemical reaction and the second biochemical reaction by the fluidic device 500, and the reaction efficiency can be doubled by the method of this embodiment of the present invention.

It will be understood by those skilled in the art that if the amount of data required for genetic information analysis is reduced and/or the proportion of the processed valid data is increased, such that the number of FOVs required to be collected for each round of sequencing reaction is reduced, i.e. the time required for image collection is reduced or the total time of biochemical reaction is increased, m reaction devices can be divided into n units by the method of the present invention, where m and n are integers greater than or equal to 1, and n is greater than or equal to twice m, so that each unit can be in different steps or stages of one round/different rounds of sequencing reaction, thereby making full use of the imaging device 102 and the fluidic device 500 and improving the reaction efficiency. It will also be appreciated by those skilled in the art that the number of units on the reaction apparatus 200 can be fully utilized to improve efficiency by using the method of the present invention in some cases contrary to the above example, such as the time required for biochemical reaction is reduced.

In some embodiments, before the sequencing reaction is performed, the sample to be sequenced, such as a DNA strand having a double-stranded or single-stranded structure, is immobilized on the surface of the channel of the first unit 41 and the second unit 42 of the reaction apparatus 200.

In the embodiment of the present invention, the repeated execution unit S112 is the second biochemical reaction-first biochemical reaction-image capturing, and means that when the sequence measurement reaction is performed on one of the units of the reaction apparatus 200, the second biochemical reaction, the first biochemical reaction, and the image capturing are sequentially performed on the sample on the unit. When the repetitive execution unit is executed a plurality of times, the method of the embodiment of the present invention may occur a repetitive execution of the first biochemical reaction-image acquisition-second biochemical reaction on the sample on the cell, and/or a repetitive execution of the image acquisition-second biochemical reaction-first biochemical reaction on the sample on the cell. It should be noted that, in general, the sequence determination reaction is performed every following cycle: the first biochemical reaction, the image acquisition, and the second biochemical reaction, enable the determination of at least one base selected from the group consisting of A, T, C, G and U. It will be understood by those skilled in the art that the definition of "repeat unit" in the present invention is merely for convenience of describing the embodiment of the present invention, and is not intended to limit the reaction sequence in the sequencing reaction.

In the embodiment of the present invention, while the sample on the first unit 41 is subjected to the second biochemical reaction and the first biochemical reaction by the fluidic device 500, the image of the sample on the second unit 42 is acquired by the imaging device 102, and then, according to the repetitive execution unit, after the second biochemical reaction and the first biochemical reaction of the sample on the first unit 41 are performed by the fluidic device 500, the image of the sample on the first unit 41 is acquired by the imaging device 102, and after the image of the sample on the second unit 42 is acquired, the second biochemical reaction and the first biochemical reaction of the sample on the second unit 42 are performed by the fluidic device 500.

In another embodiment, the sample on the first unit 41 is image-captured by the imaging device 102 while the sample on the second unit 42 is subjected to the second biochemical reaction and the first biochemical reaction by the fluidic device 500, and then, according to the repetitive execution unit, the sample on the second unit 42 is image-captured by the imaging device 102 while the sample on the first unit 41 is image-captured after the sample on the second unit 42 is subjected to the second biochemical reaction and the first biochemical reaction by the fluidic device 500, and the sample on the first unit 41 is image-captured after the sample on the first unit 41 is subjected to the second biochemical reaction and the first biochemical reaction by the fluidic device 500.

In the embodiment of the present invention, referring to fig. 14, in an initial step S111, a sample on a first unit 41 is subjected to a first biochemical reaction by using a fluidic device 500; b, acquiring an image of the sample on the first unit 41 after the first biochemical reaction by using the imaging device 102; c subjecting the sample on the second cell 42 to a first biochemical reaction using the fluidic device 500.

In another embodiment, in an initial step, a subjecting the sample on the second cell 42 to a first biochemical reaction using the fluidic device 500; b, acquiring an image of the sample on the second unit 42 after the first biochemical reaction by using the imaging device 102; c subjecting the sample on the first unit 41 to a first biochemical reaction using the fluidic device 500.

The imaging device 102 is used to perform image acquisition on the sample to form image data, which can be output to other devices/modules of the sequencing system for processing to obtain corresponding images.

In certain embodiments, step a and step c are performed simultaneously, or step b is performed before step c, or step b is performed after step c. In this way, the method of controlling sequencing is more flexible to implement.

Specifically, in the embodiment of the present invention, in step a, the sample on the second unit 42 is not affected by the first biochemical reaction of the sample on the first unit 41 when the sample on the first unit 41 is subjected to the first biochemical reaction by the fluidic device 500. And vice versa.

Preferably, steps b and c are performed simultaneously, which further increases the efficiency of the process.

In some embodiments, the first biochemical reaction comprises an extension reaction and the second biochemical reaction comprises a radical cleavage. Thus, the method for controlling the sequence determination reaction has wider application range.

Specifically, in certain embodiments, the sample to be sequenced, i.e., the template strand, has been immobilized within the channels of the first unit 41 and the second unit 42 of the reaction device 200 prior to the sequencing reaction. Polymerase/ligase extension reactions are based on base complementarity, attaching a specific substrate to the sample to be sequenced, and determining the type of substrate bound using a detectable group carried on the substrate to determine the sequence. In one example, the detectable group comprises a fluorophore that fluoresces under a laser of a particular wavelength.

The radical excision (clean) reaction is a cleavage of the radical on the substrate bound to the sample (template) to be sequenced, such that the next base of the template can continue to be determined, i.e., the sample on the first unit 41 and/or the second unit 42 can continue the sequencing reaction.

In certain embodiments, the extension reaction comprises sequencing by ligation and sequencing by synthesis.

In certain embodiments, the second biochemical reaction comprises capping.

What is called capping is primarily the group/bond that is exposed after cleavage of the protecting group. In one example, the first biochemical reaction comprises a base extension reaction, and the substrate is added in the structure of A/T/C/G-terminator-linker-luminophore, wherein the terminator is a photo-and/or chemically cleavable group, and the luminophore is carried by the substrate via linker. The second biochemical reaction involves radical cleavage, where after cleavage of the cleavable group by light and/or chemical cleavage, the exposed group is a thiol group that can be protected from oxidation by capping, e.g., by addition of an alkylating agent. Thus, the method for controlling the sequence determination reaction has wider application range.

In certain embodiments, the image acquisition further comprises adding an imaging agent. The imaging agent contains antioxidant components, such as water-soluble vitamin E (Trolox), and the like, and can avoid or reduce the damage or influence of light on a sample in the image acquisition process.

Preferably, the light emitted by the sample excited by the laser is fluorescence, so that the adverse effect of the ambient light on the image acquisition of the sample by the imaging device can be reduced.

Further illustrating: the "signal collection" process includes addition of an imaging agent, image acquisition (in embodiments of the present invention, the addition of an imaging agent is placed in the image acquisition); after clean, buffer (buffer1) wash, cap (adding some protective reagent, related to the substrate structure), and buffer2 wash (buffers 1, 2 may be the same or different).

In some embodiments, referring to fig. 13, the fluidic device 500 includes a valve body assembly 10 and a driving assembly 50, the driving assembly 50 is connected to the valve body assembly 10 through the reaction device 200, the valve body assembly 10 is used to switch different reagents when the fluidic device 500 is used to perform a first biochemical reaction and/or a second biochemical reaction on a sample in the first unit 41 and/or the second unit 42, and the driving assembly 50 enables the valve body assembly 10 to output the reagents to the first unit 41 and/or the second unit 42. In this manner, different reagents required for sequencing reactions can be conveniently input to the first unit 41 and/or the second unit 42 through the valve body assembly 10 and the driving assembly 50.

Specifically, in the embodiment of the present invention, the fluid device 500 includes a reagent assembly including a first reagent, a second reagent, and a third reagent, the reagent assembly includes a first reagent bottle 11 containing the first reagent, a second reagent bottle 12 containing the second reagent, and a third reagent bottle 13 containing the third reagent, and the valve body assembly 10 connects the first reagent bottle 11, the second reagent bottle 12, and the third reagent bottle 13 via a pipe. The valve body assembly 10 switches communication between different reagent bottles to enable the drive assembly 50 to draw reagent from a reagent bottle in communication with the valve body assembly 10 to the first unit 41 and/or the second unit 42.

In certain embodiments, the valve body assembly 10 includes a first multi-way valve 20 and a first three-way valve 30, the first multi-way valve 20 switching communication of different reagents to the first three-way valve 30, the first three-way valve 30 outputting the reagent output by the first multi-way valve 20 to the first unit 41 and/or the second unit 42. In this manner, the drive assembly 50 enables the valve body assembly 10 to output different reagents to the first unit 41 and/or the second unit 42 via the first multi-way valve 20 and the first three-way valve 30.

Specifically, in the embodiment of the present invention, the first multi-way valve 20 is connected to the first, second, and

third reagent bottles

11, 12, 13 and the first three-way valve 30 through pipes, and the first multi-way valve 20 is used to communicate the first, second, or

third reagent bottle

11, 12, 13 with the first three-way valve 30. The first three-way valve 30 is connected to the first unit 41, the second unit 42, and the first multi-way valve 20 through pipes, and the first three-way valve 30 is used for communicating the first unit 41 or the second unit 42 with the first multi-way valve 20.

In some embodiments, the first reagent is a sequencing reagent, the second reagent is a radical cleavage reagent, the third reagent is an imaging reagent, and the first multi-way valve 20 includes a first extraction port 21 connected to the first reagent bottle 11, a second extraction port 22 connected to the second reagent bottle 12, a third extraction port 23 connected to the third reagent bottle 13, and a liquid outlet 24. The liquid outlet 24 communicates with the first drawing port 21, or the second drawing port 22, or the third drawing port 23. Sequencing reagents are reagents that comprise at least a portion of the reactants of the extension reaction, for example reagents that comprise a substrate and a polymerase/ligase. The substrate carries a detectable group, for example a fluorescent group.

The first three-way valve 30 includes a liquid suction port 31, a first branch port 32, and a second branch port 33, and the liquid suction port 31 communicates with the first branch port 32 or the second branch port 33. The liquid suction port 31 communicates with the liquid outlet 24. The first unit 41 and the second unit 42 communicate with the first branch port 32 and the second branch port 33, respectively.

In the present embodiment, the first multi-way valve 20 is a rotary valve, the first extraction port 21, the second extraction port 22 and the third extraction port 23 respectively surround the liquid outlet 24, and the first extraction port 21, the second extraction port 22 and the third extraction port 23 are communicated with the liquid outlet 24 through a rotary pipe 25 rotating around the liquid outlet 24. The rotary pipeline 25 can be sequentially rotated to the positions of the first extraction port 21, the second extraction port 22 and the third extraction port 23, so that the liquid outlet 24 can be sequentially communicated with the first reagent bottle 11, the second reagent bottle 12 and the third reagent bottle 13, that is, the reaction device 200 can respectively obtain different reagents from the first reagent bottle 11, the second reagent bottle 12 and the third reagent bottle 13, and further, the sample can be subjected to a first biochemical reaction, a second biochemical reaction and image acquisition. In other embodiments, the order of communication of the liquid outlet 24 with the first extraction port 21, the second extraction port 22, and the third extraction port 23 may not be limited.

In some embodiments, when the fluid intake port 31 of the first three-way valve 30 communicates with the first branch port 32, the fluid intake port 31 is disconnected from the second branch port 33, and vice versa. The pipette port 31 may be connected to the first branch port 32 or the second branch port 33 as required for sequencing, that is, when the sample on the first unit 41 performs the second biochemical reaction and the first biochemical reaction, the first branch port 32 is connected to the pipette port 30, so that the pipette port 30 provides the required second reagent and first reagent to the first unit 41 through the first branch port 32, and after the first unit 41 finishes obtaining the second reagent and first reagent, the second branch port 33 is connected to the pipette port 31, so that the second unit 42 obtains the third reagent, and the imaging device 102 can perform image acquisition on the sample on the second unit 42.

After the sample image on the second unit 42 is collected, the second unit 42 starts to obtain the second reagent and the first reagent through the liquid suction port 31, so that the sample on the second unit 42 performs the second biochemical reaction and the first biochemical reaction, after the second unit 42 obtains the second reagent and the first reagent, the first shunt port 32 is communicated with the liquid suction port 31, the first unit 41 obtains the third reagent, and the imaging device 102 can collect the image of the sample on the first unit 41, thereby effectively reducing the time of sequence measurement and improving the efficiency of sequence measurement.

In some embodiments, the driving assembly 50 includes a first pump 51 and a second pump 52, the first pump 51 communicates with the valve body assembly 10 through the first unit 41, the second pump 52 communicates with the valve body assembly 10 through the second unit 42, the valve body assembly 10 outputs the reagent to the first unit 41 by the first pump 51, and/or the valve body assembly 10 outputs the reagent to the second unit 42 by the second pump 52 when the sample on the first unit 41 and/or the second unit 42 is subjected to the first biochemical reaction and/or the second biochemical reaction by the fluidic device 500. In this way, the reagent output by the valve body assembly 10 can be transferred to the first unit 41 and/or the second unit 42 by the first pump 51 and the second pump 52, respectively, which is convenient for operation.

Specifically, a first pump 51 and a second pump 52 respectively pipe-connect the first unit 41 and the second unit 42.

In the present example, the first pump 51 is connected to the first shunt port of the first three-way valve through the first unit 41, the second pump 52 is connected to the second shunt port of the first three-way valve through the second unit 42, when the first pump 51 is operated, the first pump 51 provides negative pressure to the first unit 41, so that the first unit 41 sequentially obtains the second reagent and the first reagent to perform the second biochemical reaction and the first biochemical reaction, after the first unit 41 obtains the second reagent and the first reagent, the first pump 51 stops providing negative pressure, the second pump 52 provides negative pressure so that the second unit 42 obtains the third reagent, and the imaging device 102 is used to perform image acquisition on the sample on the second unit 42.

It should be noted that, when the sample on the first unit 41 is subjected to the second biochemical reaction and the first biochemical reaction, the liquid outlet 24 is sequentially communicated with the second extraction port 22 and the first extraction port 21 to extract the second reagent and the first reagent, the liquid suction port 31 is communicated with the first shunt port 32, and when the first pump 51 supplies negative pressure to the first unit 41, the second reagent and the first reagent are sequentially introduced into the channel of the first unit 41.

After the first unit 41 finishes obtaining the second reagent and the first reagent, the first pump 51 stops providing negative pressure, the liquid outlet 24 communicates with the third extraction port 23 to extract the third reagent, the liquid suction port 24 communicates with the second shunt port 33, and the second pump 52 provides negative pressure to the second unit 42, so that the third reagent enters the channel of the second unit 42, and the imaging device 102 is used for carrying out image acquisition on the sample on the second unit 42. Therefore, the valve body assembly 10, the driving assembly 50 and the imaging device 102 cooperate to perform image acquisition on the sample on the second unit 42 while the sample on the first unit 41 is subjected to the second biochemical reaction and the first biochemical reaction. And vice versa.

In certain embodiments, the fluidic device 500 comprises at least one first container and a sequencing reagent dispensing assembly 60, the reagents comprising sequencing reagents, the sequencing reagent dispensing assembly 60 outputting the sequencing reagents into the first container in communication with the valve body assembly 10 when the fluidic device 500 is used to perform the first and/or second biochemical reactions on the samples on the first unit 41 and/or the second unit 42. In this manner, reagents for performing sequencing reactions are conveniently added to first unit 41 and second unit 42.

Specifically, in the present example, the first container is a first reagent bottle 11. In one example, the number of the first container is plural.

The sequencing reagent deployment assembly 60 includes a plurality of sequencing reagent feedstock bottles 61, a second multi-way valve 62, a second three-way valve 63, and a third pump 64. The plurality of sequencing reagent raw material bottles 61 are used for containing a plurality of sequencing reagent raw materials, the second multi-way valve 62 is simultaneously connected with the plurality of sequencing reagent raw material bottles 61 through pipelines, and the second three-way valve 63 is connected with the pipelines. The second three-way valve 63 also piped connects the third pump 64 and the first reagent bottle 11. The third pump 64 is connected to one of the sequencing reagent raw material bottles 61 through a second three-way valve 63 and a second multi-way valve 62. The first reagent bottle 11 is connected to the third pump 64 via the second three-way valve 63. The third pump 64 is sequentially communicated with the sequencing reagent raw material bottles 61 to extract sequencing reagent raw materials in the sequencing reagent raw material bottles 61 and mix the sequencing reagent raw materials to prepare sequencing reagents, and the third pump 64 is communicated with the first reagent bottle 11 and is used for injecting the sequencing reagents into the first reagent bottle 11.

In this embodiment, the plurality of sequencing reagent raw material bottles 61 are respectively filled with different sequencing reagent raw materials, so that the sequencing reagent raw materials in the plurality of sequencing reagent raw material bottles 61 can be sequentially extracted by the third pump 64, and then mixed and configured into a sequencing reagent.

In one example, the number of the sequencing reagent raw material bottles 61 is nine, and they contain respective solutions of different types of nucleoside analogues (substrates), a DNA polymerase solution, and components of various buffer solutions or thiol-protecting solutions. A plurality of sequencing reagent raw material bottles 61 can be placed on the test-tube rack to stabilize a plurality of sequencing reagent raw material bottles 61, can also paste respectively six sequencing reagent raw material bottles 61 simultaneously and establish different labels, so that conveniently carry out sequencing reagent raw materials next time and supply, avoid the cross infection of sequencing reagent raw materials. In other embodiments, the number of sequencing reagent raw material bottles 61 may be two, three, four, five, six, seven or eight, and other quantities, and may be specifically adjusted according to actual needs and characteristics of each solution.

The second multi-way valve 62 is configured identically to the first multi-way valve 20. The difference is that the second multi-way valve 62 realizes that the third pump 64 is communicated with the sequencing reagent raw material bottles 61 in sequence, one sequencing reagent raw material bottle 61 is selected to be communicated by the second multi-way valve 62, and the third pump 64 is controlled to adjust the extraction amount of the sequencing reagent raw material in the sequencing reagent raw material bottle 61 by controlling the communication duration. Therefore, sequencing reagent raw materials of the sequencing reagent raw material bottles 61 can be proportionally configured to meet the sequence determination requirement.

The second three-way valve 63 is provided in the same structure as the first three-way valve 30. The second three-way valve 63 may enable communication between the third pump 64 and the second multi-way valve 62, such that the third pump 64 may draw sequencing reagent raw material from the plurality of sequencing reagent raw material bottles 61 configured as sequencing reagents. The second three-way valve 63 may enable the third pump 64 to communicate with the first reagent bottle 11, so that the third pump 64 may inject the prepared sequencing reagent into the first reagent bottle 11.

The third pump 64 may supply a negative pressure to the plurality of sequencing reagent raw material bottles 61 through the second three-way valve 63 and the second multi-way valve 62 to draw the sequencing reagents in the plurality of sequencing reagent raw material bottles 61. The third pump 64 may also provide positive pressure into the first reagent bottle 11 via the second three-way valve 63 to inject sequencing reagent into the first reagent bottle 11.

Further, a first mixer 65 is connected between the second three-way valve 63 and the first reagent bottle 11, and a plurality of first serpentine pipes 651 are provided in the first mixer 65, and the plurality of first serpentine pipes 651 are connected end to end and communicated between the second three-way valve 63 and the first reagent bottle 11.

In the embodiment of the present invention, the plurality of first serpentine tubes 651 are fixed to one fixing plate, the first serpentine tubes 651 have an S-shape, and the plurality of serpentine tubes 651 may be arranged in a plurality of rows, each row communicating with each other. Utilize a plurality of first sinuous pipelines 651 to communicate between second three-way valve 63 and first reagent bottle 11 for the sequencing reagent that injects from third pump 64 buffers through a plurality of sinuous pipelines 651, increases the flow path of sequencing reagent moreover, thereby makes the abundant mixing of the multiple sequencing reagent raw materials in the sequencing reagent, promotes sequencing reagent reaction efficiency. In other embodiments, the plurality of serpentine tubes 651 can also be coiled in sequence.

The number of the first reagent bottles 11 may be one or more. In one example, the number of the first reagent bottles 11 is plural, and solutions containing different types of substrates are contained separately. The sequencing reagent configuration assembly 60 further comprises a third multi-way valve 66, wherein the third multi-way valve 66 is simultaneously connected with a plurality of first reagent bottles 11 through pipelines, the second three-way valve 63 is connected with the third multi-way valve 66 through pipelines, and a third pump 64 is communicated with one of the first reagent bottles 11 through the second three-way valve 63 and the third multi-way valve 66.

In the embodiment of the present invention, the sequencing reagents in the plurality of first reagent bottles 11 are different, and the number of the first reagent bottles 11 is four. Different sequencing reagents can be configured according to different proportions of sequencing reagent raw materials pumped by the third pump 64 from the plurality of sequencing reagent raw material bottles 61, so that a plurality of different sequencing reagents can be contained by the plurality of first reagent bottles 11. The third multi-way valve 66 is configured identically to the second multi-way valve 62. The third multi-way valve 66 may enable the third pump 64 to sequentially inject different sequencing reagents into the plurality of first reagent bottles 11, respectively. Specifically, after the third pump 64 completes the configuration of the sequencing reagent, one first reagent bottle 11 is selected through the second three-way valve 63 and the third multi-way valve 66, and the sequencing reagent is injected into the first reagent bottle 11. In other embodiments, the number of the first reagent bottles 11 may be two, three, four, five, six, or seven, and the like, and may be specifically adjusted according to actual needs and characteristics of each solution.

Further, the sequencing reagent configuration assembly 60 further includes a washing reagent bottle 67 and a first waste liquid bottle 68, the washing reagent bottle 67 is used for containing a washing reagent, the washing reagent bottle 67 is communicated with the third pump 64 through the second multi-way valve 62 and the second three-way valve 63, the first waste liquid bottle 68 is used for containing waste liquid, and the first waste liquid bottle 68 is communicated with the third pump 64 through the third multi-way valve 66 and the second three-way valve 63.

When the washing reagent bottle 67 is communicated with the third pump 64 through the second multi-way valve 62 and the second three-way valve 63, the third pump 64 can extract the washing reagent in the washing reagent bottle 67 to wash the third pump 64, namely, after one sequencing reagent is configured by the third pump 64, the washing reagent in the washing reagent bottle 67 can be firstly extracted before the sequencing reagent is configured next time, and the sequencing reagent is configured again after washing, so that cross infection in the configuration of two different gene sequencing is avoided. When the first waste liquid bottle 68 is communicated with the third pump 64 through the third multi-way valve 66 and the second three-way valve 63, the third pump 64 can inject the waste liquid which is cleaned into the first waste liquid bottle 68, thereby achieving the effect of environmental protection recovery.

In an embodiment of the invention, the sequencing reagent deployment assembly 60 performs the in-line mixing function of the fluidic device 500. It is understood that in some embodiments, the fluidic device may also be devoid of in-line mixing functionality, and accordingly, the sequencing reagent dispensing assembly 60 may be omitted, as well as being able to meet sequencing reaction fluid path requirements and control sequencing reaction fluid paths. This makes the piping of the fluid device simpler and the sequencing system smaller and more compact.

In certain embodiments, the fluidic device 500 includes a second container and an imaging reagent deployment assembly 70, the reagent including imaging reagent, the imaging reagent deployment assembly 70 outputting the imaging reagent into the second container in communication with the valve body assembly 10 upon image acquisition of the sample on the first unit 41 and/or the second unit 42 using the imaging device 102. In this manner, reagents for performing sequencing reactions are conveniently added to first unit 41 and second unit 42.

Specifically, in the present example, the second container is the third reagent bottle 13.

In an embodiment of the present invention, the imaging reagent deployment assembly 70 includes a plurality of imaging reagent feed bottles 71, a fourth multi-way valve 72, a third three-way valve 73, and a fourth pump 74. The plurality of imaging reagent material bottles 71 are configured to hold a plurality of imaging reagent materials. The fourth multi-way valve 72 simultaneously tubing connects the plurality of imaging reagent raw material bottles 71 and tubing connects the third three-way valve 73. The third three-way valve 73 also piped connects the fourth pump 74 to the third reagent bottle 13. The fourth pump 74 is in communication with one of the imaging reagent raw material bottles 71 via a third three-way valve 73 and a fourth multi-way valve 72. The third reagent bottle 13 is communicated with a fourth pump 74 through a third three-way valve 73, wherein the fourth pump 74 is sequentially communicated with the plurality of imaging reagent raw material bottles 71 to pump the imaging reagent raw materials in the plurality of imaging reagent raw material bottles 71 and mix them to prepare the imaging reagent. The fourth pump 74 is in communication with the third reagent bottle 13 for injecting imaging reagent into the third reagent bottle 13.

In the embodiment of the present invention, the plurality of imaging reagent raw material bottles 71 respectively contain different imaging reagent raw materials, so that the imaging reagent raw materials in the plurality of imaging reagent raw material bottles 71 can be sequentially pumped by the fourth pump 74, and thus mixed to prepare the imaging reagent. Specifically, the number of imaging reagent raw material bottles 71 is five. A plurality of former material bottles of formation of image reagent 71 can be placed on the test-tube rack to stabilize a plurality of former material bottles of formation of image reagent 71, can also paste respectively five former material bottles of formation of image reagent 71 and establish different labels simultaneously, in order to conveniently carry out the supplementary of formation of image reagent raw materials next time, avoid the cross infection of formation of image reagent raw materials. In other embodiments, the number of imaging reagent raw material bottles 71 may be six or eight, and the like, and is specifically adjusted according to actual needs.

The fourth multi-way valve 72 is configured identically to the first multi-way valve 20. What is different is that the fourth multi-way valve 72 realizes that the fourth pump 74 is communicated with the plurality of imaging reagent raw material bottles 71 in sequence, and the fourth multi-way valve 72 selects one of the imaging reagent raw material bottles 71 to be communicated and controls the fourth pump 74 to adjust the extraction amount of the imaging reagent raw materials in the imaging reagent raw material bottles 71 by controlling the communication duration. Therefore, the imaging reagent raw materials of the imaging reagent raw material bottles 71 can be proportionally configured to meet the sequence determination requirement.

The third three-way valve 73 is provided in the same structure as the first three-way valve 30. The third three-way valve 73 may enable communication between the fourth pump 74 and the fourth multi-way valve 72, such that the fourth pump 74 may pump imaging reagent stock from the plurality of imaging reagent stock bottles 71 configured as imaging reagent. The third three-way valve 73 may enable communication between the fourth pump 74 and the third reagent bottle 13, such that the fourth pump 74 may inject the configured imaging reagent into the imaging reagent bottle 13.

The fourth pump 74 may provide negative pressure to the plurality of imaging reagent raw material bottles 71 via the third three-way valve 73 and the fourth multi-way valve 72 to pump the imaging reagent raw material in the plurality of imaging reagent raw material bottles 71. The fourth pump 74 may also provide positive pressure into the third reagent bottle 13 via the third three-way valve 73 to inject imaging reagent into the third reagent bottle 13.

Further, the imaging reagent deployment assembly 70 further comprises a second mixer 75, the second mixer 75 being connected between the third three-way valve 73 and the third reagent bottle 13, the second mixer 75 comprising a plurality of second serpentine conduits 751, the plurality of second serpentine conduits 751 being connected end-to-end and being in communication between the third three-way valve 73 and the third reagent bottle 13.

The second mixer 75 is configured in the same manner as the first mixer 65, and the imaging agent injected from the fourth pump 74 by the second mixer 75 is buffered by the second serpentine conduits 751, and the flow path of the imaging agent is increased, so that the imaging agent raw materials in the imaging agent are sufficiently mixed, and the reaction efficiency of the imaging agent is improved.

Further, in certain embodiments, the drive assembly 50 further comprises a fourth three-way valve 53, a fifth three-way valve 54, a second waste bottle 55, and a third waste bottle 56. The fourth three-way valve 53 is piped between the first pump 51 and the first unit 41, while also being piped to a second waste bottle 55. A fifth three-way valve 54 is piped between the second pump 52 and the second unit 42, and also piped to a third waste liquid bottle 56.

The first pump 51 is connected to the first unit 41 or the second waste liquid bottle 55 through the fourth three-way valve 53, so that after the first pump 51 pumps the waste liquid in the first unit 41, which has completed the sequencing reaction, the second waste liquid bottle 55 can be injected with the waste liquid, so that the first pump 51 can provide a negative pressure to the first unit 41 for the next sequencing reaction. The fifth three-way valve 54 and the fourth three-way valve 53 are identical in structure and will not be described in detail herein, and the third waste liquid bottle 56 and the second waste liquid bottle 55 are identical in structure and will not be described herein.

In embodiments of the present invention, the imaging agent dispensing assembly 70 performs the in-line mixing function of the fluidic device 500. It will be appreciated that in some embodiments, the fluidic device may also lack in-line mixing functionality and, accordingly, the imaging agent deployment assembly 70 may be omitted. This makes the piping of the fluid device simpler and the sequencing system smaller and more compact.

In certain embodiments, the fluid device 500 includes a first control unit electrically connecting the valve body assembly 10 and the drive assembly 50 to control the operation of the valve body assembly 10 and the drive assembly 50. In this manner, automated control of the valve body assembly 10 and the drive assembly 50 may be achieved, thereby improving efficiency.

Specifically, in the present example, the first control unit electrically connects the first multi-way valve 20, the first three-way valve 30, and the driving assembly 50 to control the operation of the first multi-way valve 20, the first three-way valve 30, and the driving assembly 50. The first control unit may be a device including a single chip, a calculator, or a central control processor, and the first control unit is used to control the first multi-way valve 20, the first three-way valve 30, and the driving assembly 50 to operate, so as to realize automatic operation of the fluid device 500 and improve efficiency. Further, in the present example, the first control unit also electrically connects the second, third, fourth, third and fourth

multi-way valves

62, 63, 66, 72, 73, 64 and 74, so that the fluid device 500 is improved in operation efficiency.

In certain embodiments, a method of controlling a sequencing reaction further comprises: a plurality of set positions at which the image acquisition of the sample on the first unit 41 and/or the second unit 42 is performed with the imaging device 102 is determined. Thus, the image acquisition time of the imaging device 102 can be shortened, and the efficiency can be improved.

Specifically, the initial position at the time of image acquisition of the sample 300 of the channels of the first unit 41 and the second unit 42, for example, the initial XY position, may be input in the imaging device 102, and the distance of each movement and the number of image acquisition required for each channel are set, and the sequence determination reaction is performed from the initial position.

Generally, each unit of the reaction device 200 includes a plurality of channels to facilitate sequencing of the sample 300. The image data of the sample 300 on each channel consists of multiple fields of View (FOV). In one example, to acquire images of the sample 300 on multiple channels of the unit, 300 FOVs are set for each channel, and the moving position of the reaction apparatus 200 is controlled according to the set number of FOVs.

In some embodiments, referring to fig. 16, the imaging device 102 includes a second control unit 502, an image capturing unit 506, and a second light source 508, the second control unit 502 sends an initialization command and a driving command, the stage 103 determines a plurality of setting positions according to the initialization command, the stage 103 moves the reaction device according to the plurality of setting positions and the driving command when the imaging device 102 captures an image of the sample 300 on the first unit 41 and the second unit 42, the second control unit 502 controls the second light source 508 to emit light to the first unit 41 and/or the second unit 42 to excite the detection light when the stage 103 moves the reaction device 200 to the setting positions, and controls the image capturing unit 506 to capture the detection light to form image data. In this way, automatic control of the image acquisition of the sample 300 on the first unit 41 and the second unit 42 is achieved.

Specifically, in some embodiments, the second control unit 502 includes an upper computer 510 and a lower computer 512, and the upper computer 510 is configured to send an initialization instruction. The lower computer 512 is used for sending a driving instruction according to the initialization instruction. When the stage 103 moves the reaction apparatus 200 to a set position, the lower computer 512 is used to control the second light source 508 to emit light to the sample 300 so as to excite the sample 300 to emit detection light, and the image acquisition unit 506 is controlled to acquire the detection light to form image data. The image acquisition unit 506 is used to transmit image data directly to the upper computer 510. Therefore, the data transmission times of the upper computer 510 and the lower computer 512 can be reduced, and meanwhile, the image data is directly transmitted to the upper computer 510, so that rapid sequence determination is realized.

In some embodiments, the stage 103 directly carries the reaction device 200, the stage 103 controls the movement of the reaction device 200 in the sequence measurement system, and the stage 103 includes a position calculation unit that calculates a set position for moving the reaction device 200 each time to move the reaction device during the sequence measurement according to an initialization command. For example, in the high-throughput sequencing, image data of the sample 300 at a plurality of set positions is acquired in one sequencing, and the stage 103 calculates the set position for driving the reaction device 200 every time based on the initialization command, and moves the reaction device 200 to an area where the image acquisition unit 506 can acquire an image based on each set position when receiving the driving command. Preferably, the stage 103 can move in three XYZ and z axes to move the reaction apparatus 200 to a predetermined position.

In another embodiment, the reaction apparatus 200 may be placed on another support stage, and the stage 103 moves the reaction apparatus 200 to a set position by driving the support stage.

In certain embodiments, image acquisition unit 506 includes camera 108 to convert the optical signals into electrical signals. In one example, the image capturing unit 506 includes an optical path module and a camera 108, the reaction apparatus 200 is disposed on the stage 103 and located on an object side of the optical path module, and the camera 108 is located on an image side of the optical path module. The optical path module may be a microscope, and the microscope may include the objective lens 110 of the lens module 104 of the above embodiment.

In some embodiments, the image capture unit 506 is configured to receive an initialization command and turn on according to the initialization command. In this way, the image capturing unit 506 is in the on state after initialization, so that the speed of capturing the detection light by the image capturing unit 506 is faster.

In some embodiments, the upper computer 510 sends the initialization command to the image capturing unit 506 and receives the image data transmitted by the image capturing unit 506 through a wireless or wired method. Thus, data transmission between the upper computer 510 and the image acquisition unit 506 is realized.

Specifically, the data transmission mode between the upper computer 510 and the image capturing unit 506 may be wireless local area network transmission, bluetooth transmission, or universal serial bus transmission. Of course, in other embodiments, the method is not limited to the above-mentioned transmission method, and an appropriate transmission method may be selected according to actual requirements.

In some embodiments, the lower computer 512 includes an input/output port for outputting a first transistor-transistor logic level signal (TLL signal) to control the second light source 508 to emit light and to control the image acquisition unit 506 to acquire detected light.

Thus, the lower computer 512 controls the second light source 508 and the image capturing unit 506 through the logic level signal of the first transistor, which reduces the communication time between the lower computer 512 and the second light source 508 and the image capturing unit 506, further obtains images quickly, and realizes quick sequence measurement.

Specifically, in one example, the second light source 508 emits laser light of a specific wavelength, irradiates the sample 300 on the first unit 41 and the second unit 42, causes the fluorescent group in the sample 300 to emit fluorescence as detection light, and the image acquisition unit 506 acquires the fluorescence to form image data.

Further, the transmission rate of the transistor-transistor logic level signal is microsecond, and compared with the related art in which communication is performed through a serial port, the transistor-transistor logic level signal enables the lower computer 512 to realize fast communication with the second light source 508 and the image acquisition unit 506, so that communication time between the lower computer 512 and each component is reduced, and fast sequence determination is facilitated.

In some embodiments, when the set exposure time of the image capturing unit 506 is reached while the image capturing unit 506 captures the detection light, the second control unit 502 controls the second light source 508 to be turned off. In this way, the second control unit 502 controls the second light source 508 to emit light during the exposure time of the image capturing unit 506 and to be turned off after the exposure is finished, so that the image captured by the image capturing unit 506 is clearer and energy is saved.

Specifically, in some embodiments, the lower computer 512 controls the second light source 508 to turn off.

Further, in some embodiments, the exposure time may be set in a number of ways, such as manually set as the case may be, or a simulated exposure process may be performed prior to sequencing to obtain an optimum exposure time, or an algorithm may be used to calculate an appropriate exposure time value. Of course, in other embodiments, the exposure time is not limited to the above method, and the exposure time may be set by selecting an appropriate method according to actual conditions in practical applications.

In some embodiments, the lower computer 512 includes an input/output port for outputting a second transistor-to-transistor logic level signal to control the second light source 508 to turn off. Thus, the lower computer 512 outputs the second transistor-transistor logic level signal through the input/output port to turn off the second light source 508, so that the communication time between the lower computer 512 and the second light source 508 is reduced, and the rapid sequencing is favorably realized.

In some embodiments, after the second light source 508 is turned off, the second control unit 502 controls the stage 103 to move the reaction apparatus 200 to the next setting position to complete the acquisition of the image data of the setting position. In this way, the imaging device 102 acquires images one by one for each set position of the mobile reaction device 200, thereby realizing high-throughput sequencing.

Specifically, in some embodiments, the lower computer 512 sends the driving command to the stage 103 again after the second light source 508 is turned off. Further, when the acquisition of the image data corresponding to all the set positions is completed, the lower computer 512 is configured to send an end instruction to the upper computer 510 to complete the image acquisition of one unit of the reaction apparatus 200.

In some embodiments, the image capturing unit 506 is connected to the upper computer 510, the image capturing unit 506 transmits image data to the upper computer 510 every time it captures image data of a set position, the lower computer 512 sends a driving command to the stage 103 after the second light source 508 is turned off, so that the stage 103 moves the reaction device 200 to a next set position, and the lower computer 512 does not need to wait for the completion of image data transmission to further shorten the sequence determination time.

In some embodiments, the drive command is a pulse signal. In this way, the second control unit 502 sends the driving command to the stage 103 in the form of a pulse signal, which reduces the communication time between the second control unit 502 and the stage 103 and is beneficial to realizing rapid sequence measurement.

In some embodiments, referring to fig. 16, the image capturing unit 506 includes a focus tracking module 516 and the objective lens 110, the focus tracking module 516 controls the objective lens 110 and/or the reaction device 200 to move along the optical axis of the objective lens 110 according to the initialization command to determine an optimal focusing position when the image capturing unit 506 captures an image of the sample, and the focus tracking module 516 maintains a distance between the objective lens 110 corresponding to the optimal focusing position and the sample when the image is captured. In this way, when the set positions of the samples to be acquired are not on the same XY plane, the distance between the objective lens 110 and the reaction apparatus 200 is adjusted by the focus tracking module 516, so that the image acquisition unit 506 acquires clear images of the samples on different XY plane set positions.

Specifically, in certain embodiments, the distance of the objective lens 110 from the sample is the object distance. The upper computer 510 sends the initialization instruction to the focus tracking module 516, so that the focus tracking module 516 starts an automatic focus tracking function. In one example, movement along the optical axis of the objective lens may be understood as movement along the Z-axis.

The focus tracking module 516 controls the movement of the objective lens 110 relative to the reaction apparatus 200 according to the initialization command so that the camera 108 can obtain a clear image of the sample. After determining that the camera 108 is a clear sample image, the focus tracking module 516 performs a focus locking function, that is, when the position of the sample to be acquired changes and the distance between the objective lens 110 and the sample changes, the focus tracking module 516 compensates the change by controlling the movement of the objective lens 110, so that the sample image formed by the camera 108 is always kept clear.

The so-called best focus position corresponds to a predetermined distance between the objective lens and the sample, which may be a fixed value or a fixed range, depending on the quality of the imaging. In one example, by pre-defining quality parameters of the photo image, the best focus position can be determined by a hill-climbing search algorithm so that the quality of the image captured at the best focus position reaches the pre-set parameters.

Referring to fig. 17, an embodiment of the invention provides an optical detection system 600, the optical detection system 600 includes a control device 601, an imaging device 102 and a carrying device 100, the carrying device 100 includes a temperature control device 301 and a stage 103, the imaging device 102 includes a lens module 104, the lens module 104 includes an optical axis OP, the stage 103 is used for carrying a sample 300, the temperature control device 301 is used for adjusting the temperature of the sample 300, and the control device 601 is used for:

before image acquisition of the sample 300 by the imaging device 102 or at the time of image acquisition of the sample 300 by the imaging device 102, a range in which temperature fluctuation of the sample 300 is allowed is set by the temperature control device 301 so that a positional fluctuation range of the lens module 104 along the optical axis OP is within a preset range.

It should be noted that the explanation and description of the technical features and advantages of the imaging method in any of the above embodiments and examples are also applicable to the optical detection system 600 of the present embodiment, and are not detailed herein to avoid redundancy.

Referring to fig. 18, an embodiment of the present invention provides a sequence determination system 700 for controlling a sequence determination reaction, the sequence determination system 700 includes an optical detection system 600, the optical detection system 600 includes a control device 601, an imaging device 102 and a carrying device 100, the imaging device 102 includes a lens module 104, the lens module 104 includes an optical axis OP, the carrying device 100 includes a temperature control device 301 and a stage 103, the stage 103 is used for carrying a sample 300, the temperature control device 301 is used for adjusting a temperature of the sample 300, the control device 601 is used for performing image acquisition on the sample 300 by using the imaging device 102, and is used for:

before the sequence measurement reaction is performed by the sequence measurement system 700 or at the time of the sequence measurement by the sequence measurement system 700, a range in which the temperature fluctuation of the sample 300 is allowed is set by the temperature control device 301 so that the positional fluctuation range of the lens module 104 along the optical axis OP is within a preset range.

It should be noted that the explanation and explanation of the technical features and advantageous effects of the method for controlling a sequence determination reaction in any of the above embodiments and examples are also applicable to the sequence determination system 700 of the present embodiment, and are not detailed here to avoid redundancy.

In some embodiments, the optical detection system 600 is preset with a corresponding relationship between a temperature fluctuation range of the sample 300 and a position fluctuation range of the lens module 104 along the optical axis OP, and the control device 601 is configured to control the position fluctuation range of the lens module 104 along the optical axis OP to be within the preset range according to the corresponding relationship.

In some embodiments, the correspondence includes:

when the range of the temperature fluctuation of the sample is set to be +/-10 ℃, the position fluctuation range of the lens module along the optical axis is +/-8 microns;

when the temperature fluctuation range of the sample is set to be +/-5 ℃, the position fluctuation range of the lens module along the optical axis is +/-4 microns;

when the temperature fluctuation range of the sample is set to be +/-1.5 ℃, the position fluctuation range of the lens module along the optical axis is +/-1 micron;

when the range of the temperature fluctuation of the sample is set to be +/-0.5 ℃, the position fluctuation range of the lens module along the optical axis is +/-0.5 microns.

In some embodiments, the imaging device 102 includes a focusing module 106, the imaging device 102 is used for image acquisition of the sample 300, and the control device 601 is used for: the sample 300 is focused by the focusing module 106 and the lens module 104.

In some embodiments, the imaging device 102 is used to acquire images of the sample 300, and the control device 601 is used to: the lens module 104 is used to focus the sample 300.

In some embodiments, focusing comprises the steps of:

emitting light onto a sample 300 placed on a stage 103 by means of a focusing module 106;

moving the lens module 104 to a first setting position along the optical axis OP;

moving the lens module 104 from the first setting position to the sample 300 along the optical axis OP by a first setting step length and determining whether the focusing module 106 receives the light reflected by the sample 300;

when the focusing module 106 receives the light reflected by the sample 300, the lens module 104 moves along the optical axis OP by a second set step smaller than the first set step and uses the imaging device 102 to acquire an image of the sample 300, and determines whether the sharpness value of the image acquired by the imaging device 102 reaches a set threshold value;

when the sharpness value of the image reaches the set threshold value, the current position of the lens module 104 is saved as the saving position.

It will be appreciated that the control means 601 may be used to perform the steps associated with focusing.

In some embodiments, the focusing module 106 includes a first light source 116 and a light sensor 118, the first light source 116 is configured to emit light onto the sample 300, and the light sensor 118 is configured to receive light reflected by the sample 300.

In some embodiments, when the focusing module 106 receives light reflected by the sample 300, focusing comprises:

moving the lens module 104 along the optical axis OP toward the sample 300 by a third set step length smaller than the first set step length and larger than the second set step length, calculating a first light intensity parameter according to the light intensity of the light received by the focusing module 106, and determining whether the first light intensity parameter is larger than a first set light intensity threshold;

when the first light intensity parameter is greater than the first set light intensity threshold, the lens module 104 moves along the optical axis OP by a second set step length, and the imaging device 102 is used to collect the image of the sample 300, and whether the sharpness value of the image collected by the imaging device 102 reaches the set threshold is determined.

In some embodiments, the focusing module 106 includes two light sensors 118, the two light sensors 118 are configured to receive light reflected by the sample 300, and the first light intensity parameter is an average of light intensities of the light received by the two light sensors 118.

when the first light intensity parameter is greater than the first set light intensity threshold, the lens module 104 moves along the optical axis OP toward the sample 300 by a fourth set step that is smaller than the third set step and larger than the second set step, calculates a second light intensity parameter according to the light intensity of the light received by the focusing module 106, and determines whether the second light intensity parameter is smaller than the second set light intensity threshold;

when the second light intensity parameter is smaller than the second set light intensity threshold, the lens module 104 moves along the optical axis OP by a second set step length, and the imaging device 102 is used to collect the image of the sample 300, and whether the sharpness value of the image collected by the imaging device 102 reaches the set threshold is determined.

In some embodiments, the focusing module 106 includes two light sensors 118, the two light sensors 118 are configured to receive light reflected by the sample 300, the first light intensity parameter is an average value of light intensities of the light received by the two light sensors 118, the light intensities of the light received by the two light sensors 118 have a first difference, and the second light intensity parameter is a difference between the first difference and the set compensation value.

In some embodiments, the control device 601 is configured to: when the lens module 104 is moved by a second set step length, determining whether a first sharpness value of a pattern corresponding to a current position of the lens module 104 is greater than a second sharpness value of an image corresponding to a previous position of the lens module 104;

when the first sharpness value is greater than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is greater than the set difference, the lens module 104 is made to continue to move along the optical axis OP toward the sample 300 by a second set step length;

when the first sharpness value is greater than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is smaller than the set difference, the lens module 104 continues to move along the optical axis OP toward the sample 300 by a fifth set step smaller than the second set step so that the sharpness value of the image acquired by the imaging device 102 reaches the set threshold;

when the second sharpness value is greater than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is greater than the set difference, the lens module 104 is moved away from the sample 300 along the optical axis OP by a second set step length;

when the second sharpness value is greater than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is smaller than the set difference, the lens module 104 is moved away from the sample 300 along the optical axis OP by a fifth set step length so that the sharpness value of the image acquired by the imaging device 102 reaches the set threshold.

In some embodiments, the control device 601 is configured to: when the lens module 104 moves, determining whether the current position of the lens module 104 exceeds a second set position;

when the current position of the lens module 104 exceeds the second setting position, the lens module 104 stops moving or the focusing step stops.

In some embodiments, the carrier 100 includes:

a base plate 101;

the carrier 103 is fixed on the bottom plate, the carrier 103 is provided with a containing groove 242, the sample 300 is contained in the containing groove 242, the bottom of the containing groove 242 is provided with a through hole 2422, and the temperature control device 301 is connected with the sample 300 in the containing groove 242 through the through hole 2422;

the elastic supporting component 40 and the temperature control device 301 are elastically supported on the bottom plate 101 through the elastic supporting component 40.

In some embodiments, the elastic supporting assembly 40 includes a guiding cylinder 43 and an elastic member 44, the temperature control device 301 includes a temperature control portion 312 and a guiding column 341, the guiding column 341 is disposed on a side of the temperature control portion 312 away from the containing groove 242, the guiding cylinder 43 is fixed on the bottom plate 101, the guiding column 341 penetrates through the elastic member 44 and the guiding cylinder 43, and the elastic member 44 elastically abuts between the temperature control portion 312 and the guiding cylinder 43;

the guide cylinder 43 is a linear bearing, and the guide column 341 is in sliding contact with the balls of the linear bearing.

In some embodiments, the temperature control device 301 includes a fixing plate 321, a temperature conducting plate 322, a temperature control element 323, and a guiding column 341, the temperature control element 323 is sandwiched between the fixing plate 321 and the temperature conducting plate 322, the temperature control element 323 is in contact with the temperature conducting plate 322 and the fixing plate 321, the temperature conducting plate 322 is used for contacting the sample 300 loaded in the accommodating chamber 242, the guiding column 341 is disposed on a surface of the fixing plate 321 away from the temperature control element 323, and the guiding column 341 penetrates through the elastic supporting member 40.

In some embodiments, the temperature control device 301 further comprises a water bath chamber 324 disposed on a surface of the fixing plate 321 remote from the temperature control element 323, the water bath chamber 324 being spaced apart from the guide posts 341.

In some embodiments, the sequencing reaction comprises a first biochemical reaction and a second biochemical reaction, the first biochemical reaction and the second biochemical reaction are performed on the reaction device 200, the sequencing system 700 comprises a fluidic device 500, the fluidic device 500 is connected to the reaction device 200,

the reaction apparatus 200 includes a first unit 41 and a second unit 42, the sample 300 is placed on the first unit 41 and the second unit 42, one of repetitive execution units included in the defined sequence determination reaction is a second biochemical reaction-a first biochemical reaction-image acquisition,

the control means 601 is adapted to, after the following initial steps are completed, cause one of the first unit 41 and the second unit 42 to perform the second biochemical reaction and the first biochemical reaction of the sample 300 by using the fluidic device 500, and simultaneously, perform image acquisition of the sample 300 of the other unit by using the imaging device 102,

the initial steps include the steps of:

a subjecting the sample 300 in one of the first unit 41 and the second unit 42 to a first biochemical reaction using the fluidic device 500,

b image acquisition of the sample 300 on the unit after the first biochemical reaction is performed by the imaging device 102,

c subjecting the sample 300 on the other of the first unit 41 and the second unit 42 to a first biochemical reaction using the fluidic device 500.

In certain embodiments, step a and step c are performed simultaneously, or step b is performed before step c, or step b is performed after step c.

In some embodiments, the fluidic device 500 includes a valve body assembly 10 and a driving assembly 50, the driving assembly 50 is connected to the valve body assembly 10 through the reaction device 200, the valve body assembly 10 is used to switch different reagents when the fluidic device 500 is used to perform a first biochemical reaction and/or a second biochemical reaction on the sample 300 on the first unit 41 and/or the second unit 42, and the driving assembly 50 is used to enable the valve body assembly 10 to output the reagents to the first unit 41 and/or the second unit 42.

In certain embodiments, the valve body assembly 10 includes a first multi-way valve 20 and a first three-way valve 30, the first multi-way valve 20 switching communication of different reagents to the first three-way valve 30, the first three-way valve 30 outputting the reagent output by the first multi-way valve 20 to the first unit 41 and/or the second unit 42.

In some embodiments, the driving assembly 50 includes a first pump 51 and a second pump 52, the first pump 51 is communicated with the valve body assembly 10 through the first unit 41, the second pump 52 is communicated with the valve body assembly 10 through the second unit 42, the valve body assembly 10 is used for outputting the reagent to the first unit 41 by the first pump 51, and/or the valve body assembly 10 is used for outputting the reagent to the second unit 42 by the second pump 52 when the first biochemical reaction and/or the second biochemical reaction of the sample 300 on the first unit 41 and/or the second unit 42 is performed by the fluidic device 500.

Referring to fig. 17 and 18, a control device 601 for controlling imaging is used in an optical detection system 600, the optical detection system 600 includes an imaging device 102 and a carrying device 100, the carrying device 100 includes a temperature control device 301 and a stage 103, the imaging device 102 includes a lens module 104, the lens module 104 includes an optical axis OP, the stage 103 is used for carrying a sample 300, the temperature control device 301 is used for adjusting the temperature of the sample 300, and the control device 601 includes:

a storage device 602 for storing data, the data comprising computer executable programs;

a processor 604 for executing a computer-executable program, the executing of the computer-executable program comprising performing the method of any of the above embodiments.

In the description herein, references to the description of the terms "one embodiment," "certain embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The logic and/or steps represented in the flowcharts or otherwise described herein, such as an ordered listing of executable instructions that can be considered to implement logical functions, can be embodied in any computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. For the purposes of this description, a "computer-readable storage medium" can be any means that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: an electrical connection (electronic device) having one or more wires, a portable computer diskette (magnetic device), a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber device, and a portable compact disc read-only memory (CDROM). Additionally, the computer-readable storage medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via for instance optical scanning of the paper or other medium, then compiled, interpreted or otherwise processed in a suitable manner if necessary, and then stored in a computer memory.

In addition, each functional unit in the embodiments of the present invention may be integrated into one processing module, or each unit may exist alone physically, or two or more units are integrated into one module. The integrated module can be realized in a hardware mode, and can also be realized in a software functional module mode. The integrated module, if implemented in the form of a software functional module and sold or used as a stand-alone product, may also be stored in a computer readable storage medium.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.

Claims

1. An imaging method, wherein the method is used for an optical detection system, the optical detection system comprises an imaging device and a carrying device, the imaging device comprises a lens module, the lens module comprises an optical axis, the carrying device comprises a temperature control device and a stage, the stage is used for carrying a sample, the temperature control device is used for adjusting the temperature of the sample, and the method comprises the following steps:

2. A method of controlling a sequencing reaction, characterized in that the sequencing reaction is controlled using a sequencing system,

the sequence determination system comprises an optical detection system, the optical detection system comprises an imaging device and a bearing device, the imaging device comprises a lens module, the lens module comprises an optical axis, the bearing device comprises a temperature control device and a bearing platform, the bearing platform is used for bearing a sample, the temperature control device is used for adjusting the temperature of the sample, the sequence determination reaction comprises the step of acquiring an image of the sample by using the imaging device, and the method comprises the following steps:

3. The method according to claim 1 or 2, wherein the optical detection system is preset with a correspondence relationship between a range of allowing temperature fluctuation of the sample and a position fluctuation range of the lens module along the optical axis, and the position fluctuation range of the lens module along the optical axis is controlled to be within the preset range according to the correspondence relationship.

4. The method of claim 3, wherein the correspondence comprises:

5. The method of claim 1 or 2, wherein the imaging device comprises a focus module, and wherein image acquisition of the sample using the imaging device comprises: and focusing the sample by utilizing the focusing module and the lens module.

6. The method of claim 1 or 2, wherein image acquisition of the sample using the imaging device comprises: and tracking the sample by using the lens module.

7. The method of claim 5, wherein the focusing comprises the steps of:

emitting light onto the sample placed on the stage with the focusing module;

moving the lens module to a first set position along the optical axis;

enabling the lens module to move from the first set position to the sample along the optical axis by a first set step length and judging whether the focusing module receives the light reflected by the sample;

when the focusing module receives the light reflected by the sample, the lens module moves along the optical axis by a second set step length which is smaller than the first set step length, the imaging device is used for collecting images of the sample, and whether the sharpness value of the images collected by the imaging device reaches a set threshold value or not is judged;

and when the sharpness value of the image reaches the set threshold value, saving the current position of the lens module as a saving position.

8. The method of claim 7, wherein the focusing module comprises a light source for emitting the light onto the sample and a light sensor for receiving the light reflected by the sample.

9. The method of claim 7, wherein when the focusing module receives the light reflected by the sample, the focusing further comprises the steps of:

enabling the lens module to move to the sample along the optical axis by a third set step length which is smaller than the first set step length and larger than the second set step length, calculating a first light intensity parameter according to the light intensity of the light received by the focusing module, and judging whether the first light intensity parameter is larger than a first set light intensity threshold value or not;

and when the first light intensity parameter is larger than the first set light intensity threshold, moving the lens module along the optical axis by the second set step length, acquiring an image of the sample by using the imaging device, and judging whether the sharpness value of the image acquired by the imaging device reaches the set threshold.

10. The method of claim 9, wherein the focus module comprises two light sensors for receiving the light reflected by the sample, and the first light intensity parameter is an average of light intensities of the light received by the two light sensors.

11. The method of claim 7, wherein when the focusing module receives the light reflected by the sample, the focusing further comprises:

when the first light intensity parameter is larger than the first set light intensity threshold, the lens module moves to the sample along the optical axis by a fourth set step length which is smaller than the third set step length and larger than the second set step length, calculates a second light intensity parameter according to the light intensity of the light received by the focusing module, and judges whether the second light intensity parameter is smaller than the second set light intensity threshold;

and when the second light intensity parameter is smaller than the second set light intensity threshold, moving the lens module along the optical axis by the second set step length, acquiring an image of the sample by using the imaging device, and judging whether the sharpness value of the image acquired by the imaging device reaches the set threshold.

12. The method of claim 11, wherein the focusing module comprises two light sensors for receiving the light reflected by the sample, the first intensity parameter is an average of intensities of the light received by the two light sensors, the intensities of the light received by the two light sensors have a first difference, and the second intensity parameter is a difference between the first difference and a set compensation value.

13. A method as claimed in any one of claims 7-12, wherein when moving the lens module by the second set step, determining whether a first sharpness value of the image corresponding to a current position of the lens module is greater than a second sharpness value of the image corresponding to a previous position of the lens module;

when the first sharpness value is larger than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is larger than a set difference, enabling the lens module to continue to move along the optical axis to the sample by the second set step length;

when the first sharpness value is larger than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is smaller than the set difference, the lens module is enabled to continue to move along the optical axis to the sample in a fifth set step smaller than the second set step so that the sharpness value of the image acquired by the imaging device reaches the set threshold;

moving the lens module away from the sample along the optical axis in the second set step when the second sharpness value is greater than the first sharpness value and a sharpness difference between the second sharpness value and the first sharpness value is greater than the set difference;

when the second sharpness value is larger than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is smaller than the set difference, the lens module is moved away from the sample along the optical axis in the fifth set step size to enable the sharpness value of the image acquired by the imaging device to reach the set threshold value.

14. The method according to any one of claims 7-12, wherein when the lens module moves, determining whether the current position of the lens module exceeds a second set position;

and when the current position of the lens module exceeds the second set position, stopping moving the lens module or stopping focusing.

15. The method of claim 1 or 2, wherein the carrying means comprises:

a base plate;

the carrying platform is fixed on the bottom plate, the carrying platform is provided with a containing groove, the sample is contained in the containing groove, the bottom of the containing groove is provided with a through hole, and the temperature control device is connected with the sample in the containing groove through the through hole;

the temperature control device is elastically supported on the bottom plate through the elastic supporting component.

16. The method as claimed in claim 15, wherein the elastic support assembly includes a guide cylinder and an elastic member, the temperature control device includes a temperature control portion and a guide post, the guide post is disposed on a side of the temperature control portion away from the receiving groove, the guide cylinder is fixed on the bottom plate, the guide post penetrates the elastic member and the guide cylinder, and the elastic member elastically abuts between the temperature control portion and the guide cylinder.

17. The method of claim 16, wherein the guide cylinder is a linear bearing and the guide post is in sliding contact with balls of the linear bearing.

18. The method of claim 15, wherein the temperature control device comprises a fixing plate, a temperature conductive plate, a temperature control element and a guide post, the temperature control element is sandwiched between the fixing plate and the temperature conductive plate, the temperature control element is in contact with the temperature conductive plate and the fixing plate, the temperature conductive plate is used for contacting the sample loaded in the containing groove, the guide post is arranged on the surface of the fixing plate far away from the temperature control element, and the guide post penetrates through the elastic support component.

19. The method of claim 2, wherein the sequencing reaction comprises a first biochemical reaction and a second biochemical reaction, the first biochemical reaction and the second biochemical reaction being performed on a reaction device, the sequencing system comprising a fluidic device, the fluidic device being coupled to the reaction device,

the reaction device comprises a first unit and a second unit, the sample is arranged on the first unit and the second unit, one repeated execution unit included in the sequence determination reaction is defined as a second biochemical reaction-a first biochemical reaction-image acquisition,

the method comprising, after completion of the following initial steps, causing image acquisition of the sample of one of the first and second units by the imaging device while causing the other unit to perform the second biochemical reaction and the first biochemical reaction of the sample by the fluidic device,

the initial step comprises the steps of:

a subjecting the sample on one of the first and second cells to a first biochemical reaction using the fluidic device,

b using the imaging device to acquire images of the sample on the unit after the first biochemical reaction,

c subjecting the sample on the other of the first and second units to a first biochemical reaction using the fluidic device.

20. The method of claim 19, wherein step a and step c are performed simultaneously, or step b is performed before step c, or step b is performed after step c.

21. The method of claim 19, wherein the fluidic device comprises a valve body assembly and a driving assembly, the driving assembly is communicated with the valve body assembly through the reaction device, the valve body assembly is used for switching and communicating different reagents when the fluidic device is used for carrying out the first biochemical reaction and/or the second biochemical reaction on the sample on the first unit and/or the second unit, and the driving assembly enables the valve body assembly to output the reagents to the first unit and/or the second unit.

22. The method of claim 21, wherein the valve body assembly comprises a first multi-way valve and a first three-way valve, the first multi-way valve switching communication of the different reagents to the first three-way valve, the first three-way valve outputting the reagents output by the first multi-way valve to the first cell and/or the second cell.

23. An optical detection system, characterized in that, the optical detection system includes a control device, an imaging device and a carrying device, the carrying device includes a temperature control device and a stage, the imaging device includes a lens module, the lens module includes an optical axis, the stage is used for carrying a sample, the temperature control device is used for adjusting the temperature of the sample, the control device is used for:

24. A sequence determination system for controlling sequence determination reaction, which is characterized in that the sequence determination system comprises an optical detection system, the optical detection system comprises a control device, an imaging device and a bearing device, the imaging device comprises a lens module, the lens module comprises an optical axis, the bearing device comprises a temperature control device and a carrying platform, the carrying platform is used for bearing a sample, the temperature control device is used for adjusting the temperature of the sample, the control device is used for collecting images of the sample by using the imaging device, and is used for:

25. The system according to claim 23 or 24, wherein the optical detection system is preset with a correspondence relationship between a range of allowing temperature fluctuation of the sample and a range of position fluctuation of the lens module along the optical axis, and the control device is configured to control the range of position fluctuation of the lens module along the optical axis to be within the preset range according to the correspondence relationship.

26. The system of claim 25, wherein the correspondence comprises:

27. The system of claim 23 or 24, wherein the imaging device comprises a focusing module, wherein the imaging device is used for image acquisition of the sample, and wherein the control device is used for: and focusing the sample by utilizing the focusing module and the lens module.

28. The system of claim 23 or 24, wherein the imaging device is used for image acquisition of the sample, and the control device is used for: and tracking the sample by using the lens module.

29. The system of claim 27, wherein the focusing comprises:

emitting light onto the sample placed on the stage with the focusing module;

moving the lens module to a first set position along the optical axis;

30. The system of claim 29, wherein the focusing module comprises a light source for emitting the light onto the sample and a light sensor for receiving the light reflected by the sample.

31. The system of claim 29, wherein when the focusing module receives the light reflected by the sample, the focusing comprises:

32. The system of claim 31, wherein the focusing module comprises two light sensors for receiving the light reflected by the sample, and the first light intensity parameter is an average of light intensities of the light received by the two light sensors.

33. The system of claim 29, wherein when the focusing module receives the light reflected by the sample, the focusing comprises:

34. The system of claim 33, wherein the focusing module comprises two light sensors for receiving the light reflected by the sample, the first intensity parameter is an average of intensities of the light received by the two light sensors, the intensities of the light received by the two light sensors have a first difference, and the second intensity parameter is a difference between the first difference and a set compensation value.

35. The system of any one of claims 29-34, wherein the control device is configured to: when the lens module is moved by the second set step length, judging whether a first sharpness value of the image corresponding to the current position of the lens module is larger than a second sharpness value of the image corresponding to the previous position of the lens module;

when the first sharpness value is larger than the second sharpness value and the sharpness difference between the first sharpness value and the second sharpness value is smaller than the set difference, the lens module is made to continue to move along the optical axis to the sample in a fifth set step smaller than the second set step so that the sharpness value of the image acquired by the imaging device reaches the set threshold;

when the second sharpness value is larger than the first sharpness value and the sharpness difference between the second sharpness value and the first sharpness value is smaller than the set difference, moving the lens module away from the sample along the optical axis in the fifth set step size to enable the sharpness value of the image acquired by the imaging device to reach the set threshold value.

36. The system of any one of claims 29-34, wherein the control device is configured to: when the lens module moves, judging whether the current position of the lens module exceeds a second set position;

37. The system of claim 23 or 24, wherein the carrier means comprises:

a base plate;

38. The system of claim 37, wherein the elastic support assembly comprises a guide cylinder and an elastic member, the temperature control device comprises a temperature control portion and a guide post, the guide post is disposed on a side of the temperature control portion away from the receiving groove, the guide cylinder is fixed on the bottom plate, the guide post penetrates through the elastic member and the guide cylinder, and the elastic member elastically abuts between the temperature control portion and the guide cylinder.

39. The system of claim 38, wherein the guide cylinder is a linear bearing and the guide post is in sliding contact with a ball of the linear bearing.

40. The system of claim 37, wherein the temperature control device comprises a fixing plate, a temperature conductive plate, a temperature control element and a guiding column, the temperature control element is sandwiched between the fixing plate and the temperature conductive plate, the temperature control element is in contact with the temperature conductive plate and the fixing plate, the temperature conductive plate is used for contacting the sample loaded in the containing groove, the guiding column is arranged on the surface of the fixing plate far away from the temperature control element, and the guiding column penetrates through the elastic supporting component.

41. The system of claim 24, wherein the sequencing reaction comprises a first biochemical reaction and a second biochemical reaction, the first biochemical reaction and the second biochemical reaction being performed on a reaction device, the sequencing system comprising a fluidic device, the fluidic device coupled to the reaction device,

the control means is adapted to, after completion of the following initial steps, cause image acquisition of the sample of one of the first unit and the second unit by the imaging means while causing the other unit to perform the second biochemical reaction and the first biochemical reaction of the sample by the fluidic means,

the initial step comprises the steps of:

42. The system of claim 41, wherein step a and step c are performed simultaneously, or step b is performed before step c, or step b is performed after step c.

43. The system of claim 41, wherein the fluidic device comprises a valve body assembly and a driving assembly, the driving assembly is communicated with the valve body assembly through the reaction device, the valve body assembly is used for switching and communicating different reagents when the fluidic device is used for carrying out the first biochemical reaction and/or the second biochemical reaction on the sample on the first unit and/or the second unit, and the driving assembly enables the valve body assembly to output the reagents to the first unit and/or the second unit.

44. The system of claim 43, wherein the valve body assembly comprises a first multi-way valve and a first three-way valve, the first multi-way valve switching communication of the different reagents to the first three-way valve, the first three-way valve outputting the reagents output by the first multi-way valve to the first cell and/or the second cell.

45. A control device for an optical detection system, the optical detection system including an imaging device and a carrying device, the carrying device including a temperature control device and a stage, the imaging device including a lens module, the lens module including an optical axis, the stage for carrying a sample, the temperature control device for adjusting the temperature of the sample, the control device comprising:

a processor for executing the computer-executable program, execution of the computer-executable program comprising performing the method of any of claims 1-22.