Automatic alignment system and microscopic imaging device applied to ODT

Abstract

The embodiment of the utility model discloses an automatic alignment system applied to ODT, the automatic alignment system comprises an objective table, a first-stage lifting table and a second-stage lifting table, a scanning illumination module and the second-stage lifting table are installed on the two sides of the objective table in the Z-axis direction respectively, and the objective table is configured to bear the scanning illumination module; the objective table is slidably mounted on the first-stage lifting table, and the first-stage lifting table is configured to drive the scanning illumination module to move in the Z-axis direction; an objective lens fixing seat is mounted on the secondary lifting table, the objective lens fixing seat is used for mounting a lighting objective lens, and the secondary lifting table is configured to drive the lighting objective lens to move in the Z-axis direction.

Description

Technical Field

[0001] This utility model relates to the field of label-free microscopy imaging technology, and in particular to an automatic alignment system and microscopy imaging device for use in ODT. Background Technology

[0002] Microscopic imaging is a fundamental tool in biomedical research, commonly used for imaging biological samples, especially live cell samples. Current microscopic imaging techniques mainly include fluorescence microscopy and label-free microscopy. However, fluorescence microscopy requires specific fluorescent labels and excitation light to illuminate the sample, which can cause phototoxicity and photobleaching, making long-term live cell imaging impossible. Label-free microscopy requires optical path calibration before imaging, currently typically using manual alignment. This results in high learning costs, difficulty in operation, and low calibration accuracy in biological imaging.

[0003] Therefore, it is desirable to provide an automated alignment system and a microscopic imaging device for use in ODT, which can achieve high-precision automated alignment of the microscopic imaging device. Utility Model Content

[0004] One embodiment of this application provides an automatic alignment system for ODT, comprising: a stage, a primary lifting platform, and a secondary lifting platform, wherein a scanning illumination module and the secondary lifting platform are respectively mounted on both sides of the stage along the Z-axis, and the stage is configured to support the scanning illumination module; the stage is slidably mounted on the primary lifting platform, and the primary lifting platform is configured to drive the scanning illumination module to move along the Z-axis; an objective lens holder is mounted on the secondary lifting platform for mounting an illumination objective lens, and the secondary lifting platform is configured to drive the illumination objective lens to move along the Z-axis.

[0005] One embodiment of this application provides a microscopic imaging device, including an automatic alignment system, a scanning illumination module, and an imaging module as described in any embodiment of the present invention. The scanning illumination module illuminates the sample with rotating parallel light, which is transmitted through the sample and received by the imaging module. The imaging module generates an image of the sample. Attached Figure Description

[0006] This utility model will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting; in these embodiments, the same reference numerals denote the same structures, wherein:

[0007] Figure 1 This is an exemplary structural diagram of an automatic alignment system applied to ODT according to some embodiments of the present invention;

[0008] Figure 2 This is an exemplary structural diagram of a primary lifting platform according to some embodiments of the present utility model;

[0009] Figure 3 This is an exemplary structural diagram of a two-stage lifting platform according to some embodiments of the present utility model;

[0010] Figure 4 This is an exemplary structural diagram of a target assembly according to some embodiments of the present invention;

[0011] Figure 5 These are exemplary patterns of targets shown in some embodiments of the present invention;

[0012] Figure 6 This is an exemplary structural diagram of a liquid level sensor according to some embodiments of the present invention;

[0013] Figure 7 This is a schematic diagram showing the positional relationship between the illumination objective and the liquid surface according to some embodiments of the present invention;

[0014] Figure 8 This is an exemplary flowchart of an automatic alignment method according to some embodiments of the present invention;

[0015] Figure 9 These are exemplary structural diagrams of a microscopic imaging device according to some embodiments of the present invention;

[0016] Figure 10A This is an exemplary optical path diagram of the lighting optical path shown in some embodiments of the present invention;

[0017] Figure 10B This is a schematic diagram of the trajectory of multi-angle parallel light illumination according to some embodiments of the present invention;

[0018] Figure 11 This is a schematic diagram of the internal optical path of an imaging module according to some embodiments of the present invention;

[0019] Figure 12 This is a schematic diagram of parallel light illumination at different tilt angles according to some embodiments of the present invention;

[0020] Figure 13A This is a schematic diagram of the spectrum distribution according to some embodiments of the present invention;

[0021] Figure 13B This is a schematic diagram of the spectrum distribution according to some embodiments of the present invention;

[0022] Figure 14A This is a schematic diagram of the spectral distribution of images shown in some embodiments of the present invention;

[0023] Figure 14B This is a schematic diagram of the spectral distribution of images shown in some embodiments of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0025] Conversely, this application covers any substitutions, modifications, equivalent methods, and schemes made within the spirit and scope of this application as defined in the claims. Furthermore, to provide the public with a better understanding of this application, certain specific details are described in detail below. However, this application can be fully understood by those skilled in the art even without these detailed descriptions.

[0026] Optical diffraction tomography (ODT) is a label-free microscopic imaging technique. Refractive index is an inherent optical property of biological samples; for example, denser regions have higher refractive indices, while sparser regions have lower refractive indices. When illumination light passes through a sample, the difference in refractive index across the sample leads to variations in light propagation, resulting in a significant change in the transmitted light field relative to the illumination light field. ODT uses mathematical models to characterize the process of illumination light propagating through the material, and by actually measuring the illumination and transmitted light fields, it can deduce the refractive index distribution within the sample, thus achieving microscopic imaging.

[0027] However, the current ODT has the following problems: (1) The introduction of illumination objectives in ODT requires optical path calibration before imaging to align the spatial positions of the illumination and imaging objectives. Currently, the alignment is done manually, which is a high learning cost for bioimaging users, difficult to get started, and has low calibration accuracy; (2) It can only use fixed scanning angle and fixed mode imaging, which cannot cover imaging scenarios with different resolutions and imaging speeds; (3) ODT requires multiple images to be taken to reconstruct and calculate three-dimensional label-free microscopic images, and the shooting and calculation process is time-consuming. Users cannot obtain the reconstruction results immediately, and they are somewhat blind when searching for and selecting shooting targets, resulting in low efficiency; (4) During long-term imaging of live cells, cells may leave the imaging field of view due to cell migration or imaging field of view drift, making it impossible to image the same cell for a long time; (5) The occurrence of specific events in the life cycle of live cells (e.g., division, apoptosis, specific organelle interaction processes, etc.) is uncertain, and the event process is fleeting. If long-term rapid imaging is used, a large amount of invalid data will be accumulated, and if slow imaging is used, the event process will be missed.

[0028] To address the aforementioned problems, this utility model provides an automatic alignment system and a microscopic imaging device for ODT. The automatic alignment system includes a stage, a primary lifting stage, and a secondary lifting stage. A scanning illumination module and a secondary lifting stage are respectively mounted on both sides of the stage along the Z-axis, with the stage configured to support the scanning illumination module. The stage is slidably mounted on the primary lifting stage, which is configured to drive the scanning illumination module to move along the Z-axis. An objective lens holder is mounted on the secondary lifting stage for mounting an illumination objective lens, and the secondary lifting stage is configured to drive the illumination objective lens to move along the Z-axis.

[0029] The automatic alignment system provided in this embodiment of the invention can achieve automatic alignment between the illumination objective and the imaging objective by automatically controlling the scanning illumination module to move along the Z-axis direction through a first-stage lifting platform and automatically controlling the illumination objective to move along the Z-axis direction through a second-stage lifting platform.

[0030] Figure 1 This is an exemplary structural diagram of an automatic alignment system applied to ODT according to some embodiments of the present invention.

[0031] like Figure 1 As shown, the automatic alignment system 100 includes: a stage 110, a primary lifting platform 120, and a secondary lifting platform 130. (As shown...) Figure 1 As shown, a scanning illumination module 200 and a secondary lifting platform 130 are respectively mounted on both sides of the stage 110 along the Z-axis direction. The stage 110 is configured to support the scanning illumination module 200. When the stage 110 is placed on a horizontal plane (e.g., a plane parallel to the ground), the Z-axis direction refers to the direction perpendicular to the ground (e.g., ...). Figure 1 (Vertical direction shown). Along a direction perpendicular to the ground, a scanning illumination module 200 is mounted on one side of the stage 110, and a secondary lifting platform 130 is mounted on the other side. In some embodiments, the stage 110 is slidably mounted on a primary lifting platform 120, which is configured to drive the scanning illumination module 200 to move along the Z-axis. For example, the primary lifting platform 120 and the stage 110 are slidably connected by a vertically placed guide rail. The primary lifting platform 120 drives the stage 110 to move along the Z-axis, thereby causing the scanning illumination module 200 (and the secondary lifting platform 130) to move along the Z-axis. In some embodiments, an objective lens holder 131 is mounted on the secondary lifting platform 130 for mounting an illumination objective lens 132, and the secondary lifting platform 130 is configured to drive the illumination objective lens 132 to move along the Z-axis.

[0032] In some embodiments, the stage 110 may be a one-piece structure having a plane for supporting the scanning illumination module 200 and connection ends that are respectively connected to the primary lifting stage 120 and the secondary lifting stage 130. In some embodiments, such as Figure 1 As shown, the stage 110 can be a detachable structure, including a stage body 111 and a support body 112. A scanning illumination module 200 and a secondary lifting platform 130 are respectively installed on both sides of the stage body 111 along the Z-axis. The support body 112 connects the stage 110 and the primary lifting platform 120.

[0033] In some embodiments, the maximum displacements of the primary lifting platform 120 and the secondary lifting platform 130 are different. For example, the primary lifting platform 120 may connect to the scanning illumination module 200 and the illumination objective lens 132, and the secondary lifting platform 130 may connect to the illumination objective lens 132. The maximum displacement of the overall component (e.g., the overall structure formed by the scanning illumination module 200 and the illumination objective lens 132) is greater than the maximum displacement of the partial component (e.g., the illumination objective lens 132), therefore the maximum displacement of the primary lifting platform 120 is greater than the maximum displacement of the secondary lifting platform 130. In some embodiments, the displacement accuracy of the primary lifting platform 120 and the secondary lifting platform 130 is different. For example, to control costs, the accuracy of the motors controlling the primary lifting platform 120 and the secondary lifting platform 130 may be different.

[0034] The scanning illumination module 200 generates illumination light to scan biological samples. The illumination objective 132 illuminates the sample. Before placing the sample at the center of the stage (e.g., adding a sample), the illumination objective 132 needs to be raised to provide sufficient operating space for sample placement. After sample addition, the illumination objective 132 needs to be moved back, ensuring alignment between it and the microscope's imaging objective to guarantee optical path alignment. Automatic alignment of the illumination objective 132 and imaging objective can be achieved via a primary stage 120 and a secondary stage 130, as detailed in [link to documentation]. Figures 2-5 And its related descriptions.

[0035] In some embodiments, the scanning illumination module 200 can generate multi-angle illumination light in different modes. For example, in scenarios involving different events (such as cell division, apoptosis, migration, etc.), parallel illumination light with different scanning angles can be used for illumination. More information about the scanning illumination module 200 can be found in [link to relevant documentation]. Figures 9-10B And its related descriptions.

[0036] Figure 2 This is an exemplary structural diagram of a primary lifting platform according to some embodiments of the present invention.

[0037] Combination Figure 1 and Figure 2As shown, the first-stage lifting platform 120 includes a first guide rail 121 and a first motor 122. The first guide rail 121 is arranged along the Z-axis direction. The platform 110 (or the support body 112 of the platform 110) can be slidably installed on the first guide rail 121. The first motor 122 drives the scanning illumination module 200 to move along the Z-axis direction.

[0038] In some embodiments, the first guide rail 121 is arranged along the Z-axis direction (e.g., the sliding direction of the first guide rail 121 is parallel to the Z-axis direction), and the platform 110 is slidably mounted on the primary lifting platform 120 via the first guide rail 121. The first guide rail 121 constrains the platform 110 to move along the Z-axis direction. In some embodiments, the first guide rail 121 may include a crossed roller guide rail, which has high load-bearing capacity and small movement clearance, ensuring the safety and stability of the slidable mounting of the primary lifting platform 120 and the platform 110.

[0039] The first motor 122 can drive the stage 110 to move along the Z-axis direction on the first guide rail 121, thereby driving the scanning illumination module 200 to move along the Z-axis direction. In some embodiments, the automatic alignment system 100 includes a controller for controlling the first motor 122.

[0040] In some embodiments, the primary lifting platform 120 includes a lead screw 123, which is connected to a first motor 122 and a platform 110 (e.g., a support 112 of the platform 110). The first motor 122 includes a first wheel (not shown), which is controlled by a controller to drive the first motor 122 to rotate. The first wheel is rotatably connected to the lead screw 123 to convert the rotation of the first wheel into linear displacement of the platform 110 along the Z-axis.

[0041] In some embodiments, the lead screw 123 is a drive shaft. One end of the lead screw 123 is connected to the first motor 122 via a first rotating wheel, and the other end is connected to the stage 110. When the first motor 122 is working, it drives the first rotating wheel to rotate. The rotation of the first rotating wheel is converted into movement along the Z-axis by the lead screw 123, thereby driving the stage 110 to move along the Z-axis. That is, the lead screw 123 converts the rotation of the first rotating wheel of the first motor 122 into linear displacement of the stage 110 along the Z-axis.

[0042] In some embodiments, the first motor 122 can be a stepper motor. The stepper motor can rotate at least one preset angle in a preset direction. The preset direction refers to the direction in which the motor drives the first rotating wheel to rotate; for example, the preset direction may include clockwise and counterclockwise directions. When the motor drives the first rotating wheel to rotate in different preset directions, the lead screw 123 can drive the stage 110 and the scanning illumination module 200 to move upwards or downwards along the Z-axis. The preset angle refers to the minimum angle at which the motor drives the first rotating wheel to rotate, for example, 0.1°, 0.2°, 0.5°, 1°, 2°, 5°, 10°, 20°, 50°, etc. The stepper motor drives the first rotating wheel to rotate step by step at preset angles. The smaller the preset angle, the higher the stepping accuracy of the stepper motor. In some embodiments, the first motor 122 is a stepper motor. By reasonably setting the preset angle of the stepper motor, the stepper motor can have higher stepping accuracy, thereby improving the accuracy of the stage 110 or the scanning illumination module 200 moving along the Z-axis.

[0043] In some embodiments, the controller can control the rotation direction and rotation angle of the stepper motor.

[0044] In some embodiments, a backlash-eliminating nut is provided on the lead screw 123, which can eliminate the repeatability positioning accuracy error caused by backlash difference during the transmission of the lead screw 123. In some embodiments, the backlash-eliminating nut may be located at the end of the lead screw 123 connected to the stage 110. In some embodiments, the backlash-eliminating nut may be located at the end of the lead screw 123 connected to the first motor 122.

[0045] In some embodiments, the primary lifting platform 120 may include an encoder that communicates with the first motor 122. In some embodiments, the encoder may be a position closed-loop feedback device for the first motor 122, enabling closed-loop position control of the primary lifting platform 120 and providing micron-level accuracy for the movement of the scanning illumination module 200 along the Z-axis. For example, the rotation direction and angle of the first wheel driven by the first motor 122 can be determined by the encoder's encoded value. After determining the movement distance of the scanning illumination module 200 along the Z-axis, the operating parameters of the first motor 122 can be determined based on the rotation angle and direction, and then the first motor 122 can be controlled to operate based on these operating parameters. In some embodiments, the encoder may be a photoelectric encoder or a magnetic encoder.

[0046] In some embodiments, the movement of the primary lifting platform 120 can be controlled by a control algorithm. For example, an initial model can be trained using historical operating data of the primary lifting platform 120 to obtain a control algorithm, thereby achieving adaptive control of the primary lifting platform 120. Historical operating data of the primary lifting platform 120 includes historical position data obtained through position sensors, historical control input parameters (e.g., current and voltage of the first motor 122), and historical operation logs (e.g., historical movement commands and historical operating times). The initial model can include supervised learning models (e.g., linear regression models, support vector machine models, neural network models, etc.) and reinforcement learning models (e.g., Q-learning models, Deep Q Network models, etc.). In some embodiments, the movement of the primary lifting platform 120 can also be achieved by adjusting the control algorithm in real-time using real-time data.

[0047] In some embodiments, the primary lifting platform 120 integrates multiple sensors (e.g., image sensors, position sensors, laser sensors, etc.) to acquire data (e.g., position data, status data, etc.) of the primary lifting platform 120. In some embodiments, the data acquired by the multiple sensors can be further used for feedback control of the primary lifting platform 120 to achieve high-precision control and calibration.

[0048] In some embodiments, the primary lifting platform 120 further includes a sensor switch. The sensor switch may include a photoelectric switch. The photoelectric switch is used to scan the travel limits of the lighting module 200. The photoelectric switch can provide initial position sensing and maximum travel position sensing for the primary lifting platform 120.

[0049] Understandably, since the secondary lifting platform 130 is located on one side of the stage 110, when the primary lifting platform 120 is controlled to move the stage 110 along the Z-axis, the secondary lifting platform 130 (and the illumination objective lens 132 on the secondary lifting platform 130) will also move along the stage 110.

[0050] The automatic alignment system 100 provided in this embodiment of the invention can automatically move the scanning illumination module 200 and the secondary lifting platform 130 along the Z-axis by controlling the primary lifting platform 120. For example, controlling the primary lifting platform 120 to move the scanning illumination module 200 and the secondary lifting platform 130 upward along the Z-axis can increase the displacement of the primary lifting platform 120 and the secondary lifting platform 130, thereby providing sufficient operating space for placing the sample. In addition, since the first motor 122 of the primary lifting platform 120 is a stepper motor and is controlled by an encoder, it can provide micron-level precision for the movement of each module or component (e.g., the scanning illumination module 200, the illumination objective lens 132) along the Z-axis, thereby improving the accuracy of automatic alignment.

[0051] Figure 3 This is an exemplary structural diagram of a two-stage lifting platform according to some embodiments of the present invention.

[0052] like Figure 3 As shown, the secondary lifting platform 130 includes a second guide rail 133 and a second motor 134. The second guide rail 133 is arranged along the Z-axis direction (for example, the sliding direction of the second guide rail 133 is parallel to the Z-axis). The objective lens holder 131 is slidably mounted on the second guide rail 133. The second motor 134 drives the illumination objective lens 132 to move along the Z-axis direction.

[0053] In some embodiments, the second guide rail 133 is arranged along the Z-axis, and the objective lens holder 131 is slidably mounted on the secondary lifting platform 130 via the second guide rail 133. The second guide rail 133 constrains the objective lens holder 131 to move along the Z-axis, thereby constraining the illumination objective lens 132 to move along the Z-axis. In some embodiments, the second guide rail 133 may include a crossed roller guide rail, which has high load-bearing capacity and small movement clearance, ensuring the safety and stability of the slidable mounting of the secondary lifting platform 130 and the objective lens holder 131. In other embodiments, the second guide rail 133 may also be other types of linear guide rails, and this invention does not specifically limit this.

[0054] In some embodiments, the second motor 134 can drive the objective lens mount 131 to move along the Z-axis direction on the second guide rail 133, thereby causing the illumination objective lens 132 to move along the Z-axis direction. In some embodiments, the controller in the automatic alignment system 100 can further control the second motor 134. For example, the controller can send a control signal to the second motor 134 to make the second motor 134 work, and when the second motor 134 works, it can drive the objective lens mount 131 to move along the Z-axis direction. In some embodiments, the second motor 134 may include one of the following: a servo motor, a stepper motor, a servo motor, etc.

[0055] In some embodiments, the secondary lifting platform 130 includes a guide groove 135 and a spring 136, the objective lens holder 131 is connected to the spring 136, and the bearing of the second motor 134 is installed in the guide groove 135. A positioning groove is provided in the guide groove 135.

[0056] The guide groove 135 is used to accommodate the bearing of the second motor 134. The spring 136 is used for positioning the objective lens holder 131. One end of the spring 136 is connected to the objective lens holder 131, and the other end is connected to the stage 110 through the fixing interface 113 of the secondary lifting platform 130.

[0057] In some embodiments, the second motor 134 operates to drive the illumination objective lens 132 to move along the Z-axis. When the second motor 134 operates, the bearing rolls in the guide groove 135. When the illumination objective lens 132 moves to the designated position, the bearing of the second motor 134 is located in the positioning groove. At this time, under the pulling force of the spring 136, the objective lens holder 131 is positioned, and the illumination objective lens 132 is positioned at the designated position.

[0058] The automatic alignment system 100 provided in this embodiment of the present invention can realize the automatic movement of the illumination objective lens 132 along the Z-axis by controlling the secondary lifting platform 130. At the same time, combined with the primary lifting platform 120, the displacement along the Z-axis can be further expanded, thereby leaving sufficient operating space for placing the sample.

[0059] In some embodiments, the secondary lifting platform 130 can also be manually controlled to move the illumination objective lens 132 along the Z-axis. In this case, the structure of the secondary lifting platform 130 is similar to... Figure 3 The structure is roughly the same as the others, the difference being that the manually controlled secondary lifting platform 130 does not include a second motor, but is controlled via a knob. For example, it can be... Figure 3 The second motor is replaced by a knob. By manually rotating the knob, the rocker arm connected to the knob moves the objective lens holder 131 along the Z-axis. When the illumination objective lens 132 moves to the designated position, the bearing on the rocker arm is in the positioning groove. At this time, under the pulling force of the spring 136, the objective lens holder 131 is positioned, and the illumination objective lens 132 is positioned at the designated position.

[0060] In some embodiments, since an additional illumination objective 132 is introduced into the ODT, and multi-angle parallel light illumination is performed using the illumination objective 132, it is necessary to align the illumination objective 132 with the imaging objective. Alignment of the illumination objective 132 with the imaging objective can refer to focal plane alignment, that is, the field center of the illumination objective 132 is aligned with the field center of the imaging objective. In some embodiments, focal plane alignment of the illumination objective 132 with the imaging objective can utilize the object-image conjugate relationship, i.e., the alignment of the illumination objective 132 with the imaging objective satisfies the following conjugate relationship: the image plane of the illumination objective 132, the common object plane of the illumination objective 132 and the imaging objective, and the image plane of the imaging objective constitute a conjugate relationship. In this case, the image plane of the illumination objective 132 will be conjugate to the image plane of the imaging objective.

[0061] In some embodiments, in order to align the illumination objective 132 with the imaging objective, such as... Figure 1 As shown, the automatic alignment system 100 includes a target assembly 140. Along the Z-axis, the target assembly 140 is mounted on the scanning illumination module 200 on the side opposite the stage 110. For example, as... Figure 1As shown, the lower side of the scanning illumination module 200 is connected to the stage 110, and the target assembly 140 is mounted on the upper side of the scanning illumination module 200. In some embodiments, the target assembly 140 is configured to mount a target for automatic alignment. By adjusting the position of the target using the target assembly 140, alignment of the illumination objective lens 132 and the imaging objective lens can be achieved.

[0062] Figure 4 This is an exemplary structural diagram of a target component according to some embodiments of the present invention.

[0063] like Figure 4 As shown, the target assembly 140 includes a base 141, on which a positioning hole 142 is provided, and a target 143 is installed in the positioning hole 142. The target 143 is located at the conjugate position in the illumination optical path of the scanning illumination module 200.

[0064] The positioning hole 142 refers to the hole formed on the substrate 141. In some embodiments, the shape and size of the positioning hole 142 match the shape and size of the target 143 to allow the target 143 to be installed in the positioning hole 142. For example, both the positioning hole 142 and the target 143 are circular, and the diameter of the positioning hole 142 is not less than the diameter of the target 143. In some embodiments, the target 143 may be located at a conjugate position in the illumination optical path of the scanning illumination module 200. In this case, the state of the target 143 is called the target positioning state. The target 143 in the target positioning state can be used for automatic alignment of the illumination objective lens 132 and the imaging objective lens. In some embodiments, since the target 143 is located at a conjugate position in the illumination optical path of the scanning illumination module 200, automatic alignment of the illumination objective lens 132 and the imaging objective lens can be performed through the target 143. For example, when the target is in position, the target 143 can be photographed by the imaging objective and the camera. When the target 143 forms a clear image on the image plane of the imaging optical path and the center of the target 143 is located at the center of the image plane of the imaging optical path, the illumination objective 132 and the imaging objective are aligned.

[0065] In some embodiments, the substrate 141 is further provided with a vacancy hole 144, and the target assembly 140 includes an adjusting member 145, which is configured to adjust the position of the vacancy hole 142 and the vacancy hole 144.

[0066] In some embodiments, during bright-field imaging, uniform bright-field illumination is required for focusing and sample locating. However, when the target 143 is in its target-in-place state, it may interfere with the bright-field imaging operation. To prevent this interference, the target 143 can be moved out of its conjugate position in the illumination optical path of the scanning illumination module 200 before bright-field imaging is performed. This state, where the target 143 is moved out of its conjugate position in the illumination optical path of the scanning illumination module 200, can be considered the target-removed state. While the target 143 is in the target-removed state, bright-field imaging (focusing, sample locating, etc.) can still be performed.

[0067] The vacancy hole 144 refers to another hole opened on the substrate 141. The vacancy hole 144 is spaced apart from the positioning hole 142. When the target 143 is in the target removed state, the vacancy hole 144 is located at the conjugate position in the illumination optical path of the scanning illumination module 200, that is, the target 143 is moved out of the conjugate position in the illumination optical path of the scanning illumination module 200. Since no other structure is set in the vacancy hole 144, the illumination optical path can pass directly through the vacancy hole 144 without being affected, thereby avoiding the target 143 from interfering with bright-field imaging.

[0068] The adjusting member 145 can be used to adjust the positions of the positioning hole 142 and the empty hole 144, thereby switching the state of the target 143. For example, when automatic alignment of the illumination objective lens 132 and the imaging objective lens is required, the adjusting member 145 can be used to move the target 143 (or positioning hole 142) to the conjugate position in the illumination optical path of the scanning illumination module 200, so that the target 143 is in the target positioning state. As another example, when bright-field imaging is required (e.g., focusing, finding samples, etc.), the adjusting member 145 can be used to move the target 143 (or positioning hole 142) out of the conjugate position in the illumination optical path of the scanning illumination module 200 (that is, to move the empty hole 144 to the conjugate position in the illumination optical path of the scanning illumination module 200), so that the target 143 is in the target removed state, thereby preventing the target 143 from interfering with bright-field imaging.

[0069] In some embodiments, the adjusting member 145 includes a handle 1451 and a slide 1452, the handle 1451 being fixedly connected to the base 141, and the base 141 being slidably connected to the slide 1452.

[0070] In some embodiments, the target assembly 140 can be connected to the scanning illumination module 200 via a slide 1452. Position holes 142 and empty holes 144 are spaced apart along the extending direction of the slide 1452. An operation (such as pulling or pushing) of the handle 1451 can cause the base 141 to slide along the extending direction of the slide 1452, thereby adjusting the positions of the position holes 142 and empty holes 144, and thus switching the state of the target 143 (target removed state and target positioned state).

[0071] In some embodiments, the adjusting member 145 may also be an electric component, which can adjust the positions of the empty hole 144 and the positioning hole 142. For example, the adjusting member 145 may include a third motor, which drives the base 141 to slide along the extension direction of the slide groove 1452 to change the positions of the empty hole 144 and the positioning hole 142. The third motor is connected to the base 141 via a drive shaft. When the third motor is working, the drive shaft converts the rotation of the third motor into linear displacement of the base 141 along the extension direction of the slide groove 1452.

[0072] In some embodiments, the target assembly 140 further includes an adjustment knob 146 for adjusting the position of the target 143.

[0073] In some embodiments, when the target 143 is in the target position position, it is necessary to ensure that the center of the target 143 is aligned with the center of the field of view of the illumination optical path before subsequent alignment of the illumination objective lens 132 and the imaging objective lens can be performed. In some embodiments, the adjustment knob 146 can be disposed in the positioning hole 142. In some embodiments, the position of the target 143 can be adjusted in a plane perpendicular to the Z-axis direction (i.e., a horizontal plane parallel to the ground) using the adjustment knob 146, so that the center of the target 143 is aligned with the center of the field of view of the illumination optical path. In some embodiments, the position of the target 143 in the horizontal plane can be adjusted using the adjustment knob 146 with reference to the optical axis of the illumination optical path, so that the center of the target 143 is aligned with the center of the field of view of the illumination optical path.

[0074] In some embodiments, the pattern of the target 143 is center-oriented.

[0075] In some embodiments, the alignment of the illumination objective 132 and the imaging objective can be determined by the center of the image formed by the target 143. In some embodiments, the target assembly 140 is mounted on the side of the scanning illumination module 200 opposite to the stage 110. In this case, the target 143 mounted on the target assembly 140 is located on the image plane of the illumination optical path. When the illumination objective 132 and the imaging objective are aligned, since the target 143 is located on the image plane of the illumination optical path, the target 143 will form a clear image on the image plane of the imaging optical path, and the center of the target 143 will be located at the center of the image plane of the imaging optical path. Therefore, the target 143 can be used as an indicator of the alignment of the illumination objective 132 and the imaging objective.

[0076] Figure 5 These are exemplary patterns of targets shown according to some embodiments of the present invention. Figure 5As shown, the pattern of target 143 has a center point. The center point refers to the geometric center of the pattern of target 143. The center point has a directional function. The alignment of the illumination objective lens 132 and the imaging objective lens can be determined by using the center point of the pattern of target 143. It should be noted that the pattern of target 143 is not limited to... Figure 5 The shape shown, any other pattern with a central orientation (e.g., any graphic radiating outward from the center point) can be used as the pattern of the target 143. This utility model does not specifically limit the specific shape of the target pattern.

[0077] In ODT scenarios, samples are mostly live cells immersed in cell culture medium. Based on the principles of ODT imaging, the greater the tilt angle of the illumination light, the higher the lateral resolution of the reconstructed image. Therefore, to improve imaging resolution, a water mirror can be used as the illumination objective. A water mirror is an objective whose working medium is water. Water mirrors have a high numerical aperture (NA). During imaging, the illumination objective is immersed in the culture medium and aligned with the focal plane. Because the height of the culture medium is random, when automatically aligning the illumination objective with the focal plane, it is necessary to detect the level of the culture medium to determine the spatial relationship between the illumination objective and the focal plane, for example, whether the illumination objective is in contact with the liquid surface.

[0078] Figure 6 These are exemplary structural diagrams of liquid level sensors according to some embodiments of the present invention. Figure 6 The left image shows the illumination objective 132 separated from the liquid level sensor 1321; the right image shows the liquid level sensor 1321 installed on the illumination objective 132.

[0079] like Figure 6 As shown, in some embodiments, the automatic alignment system 100 includes a liquid level sensor 1321 mounted on the surface of the illumination objective 132, the liquid level sensor 1321 being configured to detect whether the illumination objective 132 is in contact with a liquid surface.

[0080] In some embodiments, the liquid level sensor 1321 may include an electrode sensor. The liquid level sensor 1321 is composed of a plurality of electrodes 1322 arranged in a conical shape. In some embodiments, the plurality of electrodes 1322 may be arranged around and attached to the lens surface of the illumination objective 132 at a certain spacing (equal or unequal spacing) to form a conical structure. At least a portion of the top 1323 of the illumination objective 132 is not covered by the electrodes 1322. When the illumination objective 132 (e.g., the top 1323) is not in contact with the liquid surface, the plurality of electrodes 1322 are not electrically connected to each other; when the illumination objective 132 (e.g., the top 1323) is in contact with the liquid surface, the plurality of electrodes 1322 are electrically connected simultaneously. In some embodiments, a lower-level computer can determine whether the electrodes are in a conductive or non-conductive state, thereby determining whether the illumination objective 132 is in contact with the liquid surface. Specifically, if the lower-level machine determines that the electrode is in a conductive state, it determines that the illumination objective 132 is in contact with the liquid surface; if the lower-level machine determines that the electrode is in a non-conductive state, it determines that the illumination objective 132 is not in contact with the liquid surface.

[0081] In some embodiments, electrode 1322 may be a graphite electrode. Graphite electrodes have corrosion-resistant properties, thereby extending the service life of the liquid level sensor 1321.

[0082] In some embodiments, the liquid level sensor 1321 may also include other types of sensors, such as pressure sensors. When the illumination objective 132 contacts the liquid surface, the pressure sensor is subjected to the pressure of the culture medium, and the pressure change can be measured to determine whether the illumination objective 132 is in contact with the liquid surface.

[0083] In some instances, the auto-alignment system 100 also includes a processor configured to determine whether the illumination objective 132 is in contact with the liquid surface based on the brightness of the image captured by the camera.

[0084] Figure 7 This is a schematic diagram showing the positional relationship between the illumination objective and the liquid surface according to some embodiments of the present invention.

[0085] like Figure 7 As shown, the position of the sample waveplate 710 is the initial height position, that is, the height of the sample waveplate 710 is h0 = 0. The liquid level height is h1, and the height of the illumination objective 132 is h2. The liquid level height refers to the distance between the liquid surface and the waveplate 710. The height of the illumination objective 132 refers to the shortest distance between the illumination objective 132 and the waveplate 710 along the Z-axis.

[0086] When not in focus, the height h2 of the illumination objective 132 is greater than the liquid surface height h1. The illumination objective 132 is not in contact with the liquid surface, and the brightness of the image captured by the camera changes gradually in the imaging optical path. When the illumination objective 132 moves downwards along the Z-axis to focus, its height h2 gradually decreases and approaches the liquid surface height h1 until h2 = h1. At this point, the illumination objective 132 contacts the liquid surface. The working medium of the illumination objective 132 changes from air to liquid, its converging ability increases, and the illumination intensity at the focal plane increases. In the imaging optical path, the brightness of the image captured by the camera changes abruptly. Therefore, the abrupt change in the brightness of the image captured by the camera can be used as an indicator that the illumination objective 132 is in contact with the liquid surface. For example, the processor can fit a curve based on the brightness of the image captured by the camera; the abrupt change point of the curve is the point of contact with the liquid surface, allowing for accurate determination of whether the illumination objective 132 is in contact with the liquid surface. The abrupt change point of the curve refers to the height of the illumination objective 132 where the brightness difference of the image exceeds a brightness threshold. For example, when the height of the illumination objective lens 132 decreases from the first height to the second height, the brightness difference of the image captured by the camera exceeds the brightness threshold. At this time, the point in the curve corresponding to the first height is the abrupt change point of the curve.

[0087] In some embodiments, the automatic alignment system 100 further includes a controller configured to control the movement of the primary lifting platform 120 and / or the secondary lifting platform 130 along the Z-axis.

[0088] In some embodiments, the controller can control the movement of the primary lifting platform 120 along the Z-axis, thereby controlling the movement of the scanning illumination module 200 (and the secondary lifting platform 130) along the Z-axis. In some embodiments, the controller can send a first control signal to the primary lifting platform 120 (e.g., the first motor 122) to control the movement direction (up or down) and / or movement distance of the scanning illumination module 200 along the Z-axis. The first control signal includes movement direction information and movement distance information.

[0089] In some embodiments, the controller can control the movement of the secondary lifting platform 130 along the Z-axis, thereby controlling the movement of the illumination objective lens 132 along the Z-axis. In some embodiments, the controller can send a second control signal to the secondary lifting platform 130 (e.g., the second motor 134) to control the movement direction (up or down) and / or movement distance of the illumination objective lens 132 along the Z-axis. The second control signal includes movement direction information and movement distance information.

[0090] In some embodiments, the controller may also control the third motor of the adjusting member 145 to control the state switching of the target 143. For example, the controller may send a third control signal to the third motor to control the movement direction and / or movement distance of the base 141 along the extension direction of the slide 1452.

[0091] It should be understood that the controller controlling the first-stage lifting platform 120 (e.g., the first motor 122), the controller controlling the second-stage lifting platform 130 (e.g., the second motor 134), and the controller controlling the third motor can be the same or different controllers.

[0092] In the microscopic imaging process, the sample placement and objective lens (illumination and imaging objectives) focusing are crucial steps. Sample placement involves placing the sample into the stage holder facing the objectives, allowing the stage to move the sample and achieve imaging of different fields of view. After sample placement, objective lens focusing is required. In the ODT imaging optical path, because there are two objectives—illumination and imaging—an imaging process includes focusing both objectives. Furthermore, to ensure consistent fields of view, the centers of the two objectives must be aligned. This embodiment of the invention provides an automatic alignment method for ODT, see details below. Figure 8 And related content.

[0093] Figure 8 This is an exemplary flowchart of an automatic alignment method according to some embodiments of the present invention.

[0094] In some embodiments, process 800 may be executed by a controller or processor on the automatic alignment system 100 shown in some embodiments of this invention. For example... Figure 8 As shown, process 800 includes:

[0095] Step 810: Control the primary and secondary lifting platforms to raise the illumination objective to its furthest point from the imaging objective. In some embodiments, step 810 can be performed by a controller controlling the primary lifting platform 120 and the secondary lifting platform 130.

[0096] In some embodiments, the controller can move the primary lifting platform 120 upwards along the Z-axis to its highest position, and control the secondary lifting platform 130 to move upwards along the Z-axis to its highest position. At this point, the illumination objective lens reaches its furthest point from the imaging objective lens. Combined with... Figure 1 and Figure 2 As shown, the highest point of the first-stage lifting platform 120 moving upward along the Z-axis refers to the stage 110 (or support 112) moving upward along the sliding direction of the first guide rail 121 to the upper limit position of the first guide rail 121. At this time, the stage 110 drives the scanning illumination module 200 and the illumination objective lens 132 to move upward to the maximum displacement of the first-stage lifting platform 120. Combined with... Figure 1 and Figure 3As shown, the highest point of the secondary lifting platform 130 moving upward along the Z-axis direction refers to the objective lens holder 131 moving upward along the sliding direction of the second guide rail 133 to the upper limit position of the second guide rail 133. At this time, the objective lens holder 131 drives the illumination objective lens 132 to move upward to the maximum displacement of the secondary lifting platform 130. In some embodiments, in order to accelerate the automatic alignment process, the controller can simultaneously control the movement of the primary lifting platform 120 and the secondary lifting platform 130. In some embodiments, the movement of the primary lifting platform 120 and the secondary lifting platform 130 can also be controlled separately, and the order of control is not limited.

[0097] In some embodiments, when performing microscopic imaging, the illumination objective must first be reset, i.e., raised upwards along the Z-axis to its furthest point from the imaging objective. At this point, the microscope stage and the illumination objective have the maximum operating space, facilitating sample placement, sample rack placement, and water replenishment in the live cell workstation. The live cell workstation is a device used to maintain a stable and humid environment for live cell samples during long-term imaging. Some live cell workstations require water injection to maintain sample environmental humidity and prevent evaporation of the live cell culture medium.

[0098] Step 820: Align the sample at the center of the stage with the imaging objective.

[0099] Place the sample, carried in a culture dish, into the center of the stage. In some embodiments, the type of objective lens can be determined as needed, and the corresponding subsequent operations can be performed accordingly. For example, if the objective lens is a water immersion lens, water is added to the culture dish; if the objective lens is an oil immersion lens, oil is added to the culture dish; if the objective lens is an air immersion lens, no working medium needs to be added. After determining the objective lens type and completing the corresponding operations, align the sample at the center of the stage with the imaging objective lens, and install imaging accessories such as sample clips and live cell workstation covers.

[0100] Step 830: Use the imaging objective lens on the microscope frame to locate the focal plane where the cells in the culture dish are located. This step is a standard procedure in microscope operation and will not be described in detail.

[0101] Step 840: Switch the target state to the target in position state. In some embodiments, step 840 can be performed by the controller controlling the target component 140.

[0102] In some embodiments, the target state can be adjusted to the target position state by controlling the adjustment element 145 (such as a third motor) of the target assembly 140. The target serves as a reference for aligning the imaging objective and the illumination objective. When the center of the target is located at the center of the image plane of the imaging optical path, the illumination objective and the imaging objective are aligned.

[0103] Step 850: Control the primary lifting platform to move the illumination objective lens to the working position. In some embodiments, step 850 can be performed by the primary lifting platform 120 controlled by a controller.

[0104] In some embodiments, since the secondary lifting stage 130 does not have micron-level lifting accuracy, when aligning the illumination objective and the imaging objective, the controller can first control the secondary lifting stage 130 to descend to its lowest point along the Z-axis, thereby significantly reducing the distance between the illumination objective and the imaging objective. Then, the controller controls the primary lifting stage 120 to move along the Z-axis to adjust the position of the illumination objective with micron-level lifting accuracy, precisely moving it to the working position.

[0105] In some embodiments, the working position can be obtained through calibration. The calibration process is as follows: Let the working distance of the imaging objective be d1, the working distance of the illumination objective be d2, and the sample waveplate thickness be d3. Based on the confocal property of the illumination and imaging objectives, the distance d between the illumination objective and the imaging objective is d = d1 + d2 + d3. The working distance of the imaging objective refers to the distance between the front lens surface of the imaging objective and the sample surface. The working distance of the illumination objective refers to the distance between the front lens surface of the illumination objective and the sample surface. When the imaging objective is aligned and in the working position, the working position of the illumination objective is set to a position at a distance d from the imaging objective. That is, when the illumination objective moves to the working position, the distance between the front lens surface of the illumination objective and the front lens surface of the imaging objective is d.

[0106] In some embodiments, when performing multiple OTD imaging operations, it may be necessary to change the sample for imaging. During the initial operation, steps 810 to 850 have completed the alignment of the illumination objective and the imaging objective, as well as the adjustment of the field of view center. If a sample needs to be changed, the primary and secondary lifting platforms can be controlled to move away from the working position, raising the illumination objective to its furthest point away from the imaging objective. After the sample change is completed, the primary and secondary lifting platforms are controlled to automatically move back to the memorized working position. During the sample change process, because it only involves the movement of the lifting platforms along the Z-axis and the time is short, it has high position repeatability. Therefore, a secondary imaging experiment can be performed, and this cycle can be repeated to complete subsequent sample imaging. When the user determines from the image that the field of view center has shifted, the alignment process of steps 810 to 850 can be restarted.

[0107] The automatic alignment method provided in this embodiment of the invention can automatically align the illumination objective and the imaging objective, which reduces the learning cost for operators and improves calibration accuracy due to the micron-level lifting precision of the primary lifting stage. Furthermore, the process 800 has high repeatability, improving imaging efficiency and throughput, making it suitable for high-throughput biological sample imaging scenarios requiring a large number of samples.

[0108] It should be noted that the above description of process 800 is merely for illustration and explanation, and does not limit the scope of application of this utility model. Those skilled in the art can make various modifications and changes to process 800 under the guidance of this utility model.

[0109] In some embodiments, after the automatic alignment method in process 800, subsequent ODT imaging acquisition and image reconstruction can be performed. In some embodiments, after the automatic alignment process is completed, the control module of the microscopic imaging device (e.g., control module 920 below) can control the number of scanning angles and / or illumination angles of the scanning illumination module 200 (e.g., scanning galvanometer 108 below) to achieve parallel light illumination at different tilt angles. Then, the sample is photographed using a camera. In one shooting cycle, the camera can take N images (N can be in the range of 6 to 360 images). The field-programmable gate array outputs two analog modulation signals to control the two scanning galvanometers to rotate following the camera's shooting. For each image captured by the camera, the direction of the illumination light output by the scanning galvanometer rotates 360° / N, and the camera outputs a synchronous trigger signal to the acousto-optic modulator to control the laser output. After the camera captures the image, it can be transmitted to a computer via a communication line (e.g., Camera Link or Coxpress), and the computer calls the control algorithm to perform image reconstruction. In some embodiments, the three-dimensional refractive index distribution can be reconstructed based on control algorithms (e.g., Retov approximation, Wiener reconstruction). For more information on imaging acquisition and image reconstruction, please refer to [link to relevant documentation]. Figures 9-14B And related content.

[0110] In some embodiments, after step 850, with the illumination objective in the working position, the culture medium in the cell culture dish may be higher or lower than the working height of the illumination objective, depending on the operating habits of different users. Therefore, to accommodate different user operating habits, the automatic alignment method may also include liquid level detection. In some embodiments, liquid level detection may be performed by a liquid level sensor. In some embodiments, liquid level detection may include the following steps:

[0111] Step 1: Use a liquid level sensor to detect whether the height of the illumination objective lens is lower than the liquid surface, and obtain the detection result.

[0112] In some embodiments, the liquid level sensor may include multiple electrodes (such as...) Figure 6 The electrode sensor shown can detect whether the height of the illumination objective is below the liquid surface based on the conductivity of the electrode. When the electrode is in the conductive state, it indicates that the illumination objective is in contact with the liquid surface, and the detection result is that the height of the illumination objective is below the liquid surface. When the electrode is in the non-conductive state, it indicates that the illumination objective is not in contact with the liquid surface, and the detection result is that the height of the illumination objective is not below the liquid surface.

[0113] Step 2: If the detection result shows that the height of the illumination objective is lower than the liquid surface, then the illumination objective is deemed to meet the working conditions.

[0114] As mentioned earlier, during imaging operations, the illumination objective needs to be immersed in the culture medium to align with the focal plane. That is, the illumination objective's operating condition is that its height is below the liquid surface. Therefore, when the detection result in step 1 indicates that the illumination objective's height is below the liquid surface, the illumination objective meets the operating conditions. At this point, the liquid level sensor can send a signal to the processor, determining the illumination objective's height as the liquid level.

[0115] Step 3: If the detection result shows that the height of the illumination objective is not lower than the liquid level, then it is determined that the illumination objective does not meet the working conditions.

[0116] When the illumination objective does not meet the working conditions, it is necessary to search for the liquid level a second time, and control the illumination objective to move downward along the Z-axis through the primary lifting platform to determine whether the height of the illumination objective after descending is lower than the liquid level.

[0117] In some embodiments, when the illumination objective does not meet the working conditions, the primary lifting platform can be controlled to descend a preset distance, thereby causing the illumination objective to descend a preset distance along the Z-axis. The liquid level sensor then detects again whether the height of the descended illumination objective is lower than the liquid level. In some embodiments, to ensure that the descended illumination objective does not collide with the imaging objective and cause damage, the preset distance can be less than the working distance d2 of the illumination objective.

[0118] When the height of the descending illumination objective is lower than the liquid surface, the descending illumination objective meets the working conditions. At this time, the first-stage lifting platform can be controlled to stop moving and the first-stage lifting platform can be controlled to move the illumination objective to the working position.

[0119] If the height of the descending illumination objective is still not below the liquid surface, then the descending illumination objective does not meet the working conditions. Feedback can be generated to indicate that the current sample does not meet the requirements and needs to be replaced.

[0120] In some embodiments, due to differences in the culture dishes or issues with objective lens positioning accuracy, the working position of the illumination objective obtained in step 850 may not be the optimal working position. Therefore, a second, precise search for the optimal working position of the illumination objective is required. The optimal working position is also called the optimal focal plane position. When the illumination objective is in the optimal focal plane position, the imaging objective achieves precise focusing with the illumination objective.

[0121] In some embodiments, the automatic alignment method further includes controlling the primary lifting platform to move within a preset range of the working position by step distance; determining the optimal focal plane position based on the curve relationship between the target image sharpness and the step distance; and controlling the primary lifting platform to drive the illumination objective lens to move to the optimal focal plane position.

[0122] The preset range refers to a pre-defined distance range. Since the first motor of the primary lifting platform can be a stepper motor, the stepper motor allows the primary lifting platform to move along the Z-axis in step distances. Controlling the primary lifting platform to move once within the preset range of the working position in step distances allows the camera to capture a target image and send it to the processor. The processor analyzes and processes the sharpness of the target image and the step distance, generating a curve relating the sharpness of the target image to the step distance. The optimal focal plane position is determined by fitting the curve. In some embodiments, the optimal focal plane position can be determined based on feature points (e.g., extreme points, inflection points) in the fitted curve. For example, when the sharpness of the target image corresponding to a certain step distance in the fitted curve is a maximum value, the position reached by the primary lifting platform moving that step distance is the optimal focal plane position. In some embodiments, after determining the optimal focal plane position, the primary lifting platform can be controlled to drive the illumination objective lens to move to the optimal focal plane position.

[0123] In some embodiments, after the illumination objective lens moves to the optimal focal plane position, the target image can be captured again at the current position. The host computer determines whether the found position is the optimal focal plane position based on the captured target image. If it is, the automatic alignment process is completed. If it is not the optimal focal plane position, the search range can be expanded for another search. For example, the optimal focal plane position can be searched within a second preset range, which is larger than the preset range. The search process repeats the above steps, that is, controlling the primary lifting platform to move within the second preset range of the working position by step distance; determining the optimal focal plane position based on the curve relationship between the target image sharpness and the step distance; and controlling the primary lifting platform to drive the illumination objective lens to move to the optimal focal plane position.

[0124] Finding the optimal focal plane position using the methods described above can improve calibration accuracy.

[0125] This invention also provides a microscopic imaging device, including an automatic alignment system 100, a scanning illumination module 200, and an imaging module. The automatic alignment system 100 is used to automatically align the illumination objective and the imaging objective. The scanning illumination module illuminates the sample with rotating parallel light, which is transmitted through the sample and received by the imaging module, which then generates an image of the sample.

[0126] Figure 9 This is an exemplary structural diagram of a microscopic imaging device according to some embodiments of the present invention.

[0127] like Figure 9 As shown, the microscopic imaging apparatus 900 includes an automatic alignment system 100, a scanning illumination module 200, and an imaging module 910. In some embodiments, the automatic alignment system 100 and the scanning illumination module 200 are mounted on a microscope frame 901, and the automatic alignment system 100 is used for the automatic alignment of the illumination objective and the imaging objective on the microscope frame.

[0128] Figure 10A This is an exemplary optical path diagram of the lighting optical path shown in some embodiments of the present invention.

[0129] Reference Figure 10A The dashed arrows indicate the LED lighting optical path, in which LED light source 101 serves as the light source for this optical path; the solid arrows indicate the ODT lighting optical path, in which ODT laser light source 109 serves as the light source for this optical path.

[0130] In some embodiments, the scanning illumination module 200 can provide multi-angle parallel light illumination to the sample. In some embodiments, the scanning illumination module 200 may include an LED light source 101 and a scanning optical path assembly. The scanning optical path assembly includes optical elements such as a scanning galvanometer 108, a tube mirror 104, and a dichroic mirror 103. The illumination objective 132 is immersed below the liquid surface in the culture dish 106, and a target 143 is used for automatic alignment of the illumination objective 132 and the imaging objective 107. The dichroic mirror 103 is used to couple the illumination light from the LED light source 101 and the illumination light from the ODT laser light source 109 into the tube mirror 104. The illumination objective 132 and the tube mirror 104 can form a 4F relationship. The 4F relationship refers to the alignment of the focal points of the front focal plane of the illumination objective 132, the rear focal plane of the illumination objective 132, the front focal plane of the tube mirror 104, and the rear focal plane of the tube mirror 104. At this point, the object plane of the illumination objective 132 and the image plane of the tube mirror 104 are conjugate. The scanning galvanometer 108 is placed on the image plane of the tube mirror 104, and the ODT laser source 109 illuminates the scanning galvanometer 108, which reflects the light as tilted parallel light. Because the scanning galvanometer 108 and the object plane of the illumination objective 132 are conjugate, tilted parallel light is formed on the object plane of the illumination objective 132. In some embodiments, the angle of the scanning galvanometer 108 is proportional to the control voltage. Therefore, different tilt angles of the scanning galvanometer 108 can be achieved by applying different voltages, thereby achieving parallel light illumination of the illumination objective 132 at different angles.

[0131] In some embodiments, the maximum illumination angle of the parallel light is limited by the numerical aperture NA of the illumination objective. obj For example, the numerical aperture of the illumination objective is NA. obj The system consisting of a tube endoscope and an illumination objective lens has a magnification of M. ill The illumination angle M of the tilted parallel light formed by the scanning galvanometer is... ill ≤NA obj .

[0132] Figure 10B This is a schematic diagram of the trajectory of multi-angle parallel light illumination according to some embodiments of the present invention.

[0133] In the ODT imaging system, the scanning galvanometer 108 enables parallel illumination light to be scanned 360° along the generatrix of the conical surface. For example... Figure 10B As shown, and These represent the wave vector squares of the i-th and (i+1)-th optical lines scanned along the conical surface, respectively. For the scanning galvanometer 108, a sinusoidal voltage with a phase difference of π / 2 is applied to the X-axis and Y-axis of the scanning galvanometer 108, respectively, as a function of time. The envelope of the emitted light from the scanning galvanometer 108 forms a conical surface with an angle of β, where β is proportional to E0. At small angles (e.g., 0° to 5°), the sine curve forms a circular scanning trajectory with a radius of r = fβ at a distance f from the sample. On the object plane of the illumination objective, a tilted scanning light along the conical surface with an angle β′ = Mβ is formed, where M is the magnification of the illumination optical path system.

[0134] In some embodiments, the imaging module 910 includes an imaging objective, a microscope frame 901, and a camera. The imaging objective and microscope frame 901 together form a microscopic imaging system. The scanning illumination module 200 illuminates the sample with rotating parallel light, which is received by the imaging objective after passing through the sample. This light is then relayed and amplified in subsequent optical paths and coherently imaged on the camera surface, generating a sample image. In some embodiments, off-axis holography can be used for interferometric imaging, and the phase distribution of the parallel illumination light in each direction after passing through the sample can be calculated from the interference image. In some embodiments, a reference light can be introduced into the optical path. On the camera target surface, the reference light interferes with the illumination light transmitted through the sample, and the phase distribution is obtained through interference fringes. In some embodiments, the reference light and the illumination light can be from the same source to ensure that they satisfy the phase interference condition. For example, the reference light and the illumination light can be emitted from the same laser and obtained by beam splitting.

[0135] Figure 11 This is a schematic diagram of the internal optical path of an imaging module according to some embodiments of the present invention.

[0136] like Figure 11 As shown, the dashed line represents the illumination light carrying sample information, and the solid line represents the reference light. The illumination light carrying sample phase information is incident into the imaging module 910, i.e., the illumination light 1101. The first lens 1102 and the second lens 1103 form a relay optical path, so that the illumination light 1101 is focused onto the target surface of the camera 1105. Based on the imaging principle of ODT, the illumination light 1101 carrying sample information (i.e., phase information) cannot be directly detected by the camera 1105, so an off-axis holographic method is used to obtain the phase information. In the imaging module 910, a tilted illumination reference light is introduced. The laser pigtail 1107 outputs laser light, which serves as the reference light. The reference light is collimated by the third lens 1106 and then reflected by the semi-reflective mirror 1104. It interferes with the illumination light on the target surface of the camera 1105, and then the phase distribution information of the sample is calculated based on the off-axis holographic algorithm.

[0137] In some embodiments, reference Figure 9 The microscopic imaging device 900 also includes a control module 920 for controlling the scanning angle of the scanning galvanometer 108 in the scanning illumination module 200, or controlling the number of illumination angles of the illumination module 200.

[0138] In some embodiments, the control module 920 can control the scanning angle of the scanning mirror 108 in the scanning module 200 to achieve parallel light illumination at different tilt angles, thereby adjusting the axial and lateral resolutions of the ODT reconstructed image.

[0139] Figure 12 This is a schematic diagram of parallel light illumination at different tilt angles according to some embodiments of the present invention. Figure 12 The diagram illustrates parallel light illumination at three different tilt angles, namely Light Angle 0, Light Angle 1, and Light Angle 2. In some embodiments, the rotation angle of the scanning galvanometer 108 can be controlled by the control module 920 to obtain parallel light at these three different tilt angles.

[0140] In some embodiments, combined with Figure 10A , Figure 10B and Figure 12 The illumination objective lens 132 and the tube lens 104 form a 4F relationship. At this time, the incident light is emitted and then becomes an outgoing light with an angle θ. Due to the conjugate relationship, an angle θ′=Mθ will be formed on the object surface to form a scanning tilted light along the conical surface, where M is the magnification of the illumination optical path system.

[0141] like Figure 10B As shown, the wave vector of the sample illuminated by the tilted illumination light is k0. After the tilted illumination light passes through the sample, it will obtain a wave vector k of the sample. 0x Direction and k 0z The phase distribution of the direction-modulated light, with its tilted illumination, is equivalent to a frequency-shifting operation on the phase distribution of the sample. During ODT reconstruction, the frequency-shifted phase needs to be restored and stacked across multiple directions. During restoration, the spectrum is adjusted according to (-k... 0x ,-k 0z ) The frequency is shifted in the opposite direction, thus obtaining the following: Figure 13A and Figure 13B The spectral distribution shown.

[0142] Figure 13A This is a schematic diagram of the spectrum distribution according to some embodiments of the present invention; Figure 13B This is a schematic diagram of the spectrum distribution according to some embodiments of the present invention. Figure 13A and 13B k is the spectrum reconstructed by ODT x -k z Cross-sectional schematic diagram. Wherein, Figure 13A It is a spectral distribution with a small tilt angle. Figure 13B It is a spectral distribution with a large tilt angle. In some embodiments, k 0x With k 0z The magnitude of the component can determine the spectrum k x With kz The cutoff frequency of the axis affects both axial and lateral resolution. Specifically, the smaller the tilt angle of the illumination light, the lower the k-frequency. z The higher the cutoff frequency of the axis, the higher the axial resolution of the ODT, and the higher the k-axis resolution. x The lower the axis cutoff frequency, the lower the lateral resolution of the ODT. Conversely, the larger the tilt angle of the illumination light, the lower the k-axis resolution. z The lower the axis cutoff frequency, the lower the axial resolution of the ODT, k x The higher the axis cutoff frequency, the higher the lateral resolution of the ODT.

[0143] Therefore, the scanning angle of the scanning galvanometer 108 can be controlled by the control module 920 to achieve parallel light illumination at different tilt angles, thereby adjusting the axial and lateral resolution of the ODT reconstructed image.

[0144] In some embodiments, the scanning illumination module 200 can generate a tilted light illumination sample that is rotated and scanned along a conical surface (e.g., Figure 10B As shown, by acquiring the phase distribution map of the sample after the plane wave passes through the sample from multiple different angles, the three-dimensional refractive index distribution of the sample is solved by jointly solving the corresponding multiple phase images.

[0145] ODT imaging acquisition can capture N images in one shooting cycle. In some embodiments, N can be in the range of 6 to 360 images. The larger N is, the more images are captured, and the higher the reconstruction resolution; the smaller N is, the fewer images are captured, and the lower the reconstruction resolution.

[0146] Figure 14A This is a schematic diagram of the spectral distribution of images shown in some embodiments of the present invention. Figure 14B This is a schematic diagram of the spectral distribution of images shown in some embodiments of the present invention. Figure 14A and 14B It is the spectral distribution of the image obtained based on different numbers of illumination angles. Figure 14A and 14B k is the spectrum reconstructed by ODT x -k y Cross-sectional schematic diagram. Wherein, Figure 14A It was obtained based on 30 lighting angles. Figure 14B This is based on 60 lighting angles. (Comparison) Figure 14A and Figure 14B It can be seen that, Figure 14B The spectral distribution of the image has higher resolution. The more illumination angles there are, the higher the resolution of the reconstructed image; conversely, the fewer illumination angles there are, the lower the resolution of the reconstructed image.

[0147] In some embodiments, a wider illumination angle means a slower imaging speed. Therefore, to meet different needs in different scenarios, ODT imaging can be divided into multiple modes according to the operating conditions.

[0148] Mode 1 is used for rapid reconstruction, enabling label-free 3D imaging and fast image preview, allowing operators to obtain reconstruction results in real time. In this mode, the number of illumination angles can be less than 30 degrees to ensure fast imaging speed.

[0149] Mode 2 is used for long-term image sequence capture, providing a moderate amount of data while maintaining resolution. In this mode, the number of illumination angles ranges from 30 to 120 degrees to balance imaging speed and resolution, achieving a moderate imaging speed, moderate resolution, and moderate data volume.

[0150] Mode 3 is for scenarios requiring high-resolution imaging. In this mode, the number of illumination angles is greater than 120 to ensure high resolution.

[0151] The microscopic imaging device provided in this embodiment of the invention can meet different needs in different scenarios by setting multiple imaging modes, thereby improving the flexibility of use. For example, if you want to image a cell division event, you can use mode three to image when the cell division event is about to occur, and use mode one to image at other times. This can accurately reconstruct the event process and avoid accumulating a large amount of invalid data, thus improving efficiency.

[0152] This invention provides a label-free real-time microscopic imaging method. The method enables label-free real-time microscopic image acquisition and reconstruction, and the process is as follows: A scanning illumination module generates parallel illumination light rotating 360°. The camera acquires N images from different illumination directions according to a trigger signal, which are transmitted via a high-speed camera data cable to a circular buffer in computer memory. Subsequently, an image acquisition thread retrieves image sequences from each direction from the circular buffer. For each image sequence, a reconstruction thread transmits it to a GPU, where holographic phase calculation, parameter estimation, and spectral fusion are performed sequentially. After spectral fusion of all image sequences, the reconstruction thread enhances the image quality of the reconstructed image. Finally, the label-free 3D tomographic reconstructed image is transmitted to computer memory for image display and further processing and analysis.

[0153] This invention provides a label-free three-dimensional volume tracking method for live cells. This method can identify specific cells or structures, calculate their spatial position, and use a planar electric displacement stage and a Z-axis lifting stage for position compensation to lock the cell's location. Cell tracking based on fluorescence imaging introduces additional phototoxicity, leading to cell death and preventing long-term tracking. Imaging methods based on bright-field imaging or DIC imaging can only perform two-dimensional imaging, failing to recognize changes in the cell along the Z-axis and offering low imaging resolution. The label-free real-time microscopy imaging method provided by this invention can acquire the three-dimensional refractive index distribution of cells in real time without labeling. Within a single imaging field, it can obtain the cell's positional distribution in three-dimensional space with higher resolution. Through image recognition methods and motion compensation in three-dimensional space, live cell three-dimensional volume tracking can be achieved. The label-free imaging method does not introduce phototoxicity and does not affect the normal cell life cycle; therefore, it is suitable for imaging organoids, embryos, and other organisms that are sensitive to phototoxicity.

[0154] This invention provides a label-free event-triggered imaging method. In live-cell imaging, the occurrence of specific events has a certain degree of randomness. When microscope throughput is limited, the microscope can only observe a limited area within the same time period. Continuous observation of multiple areas would generate a large amount of redundant data, and combined with fluorescence imaging methods, it is not suitable for long-term, continuous observation of live cells. The label-free real-time microscopy imaging method provides a non-phototoxic real-time detection method for live cells, offering more intelligent and flexible applications in online decision-making, event-triggered imaging, and intelligent imaging parameter control.

[0155] In long-term live-cell imaging, the imaging system acquires label-free tomographic images of cells or organelles in real time and performs online image analysis on user-specified channels or regions to monitor key events, thus constructing different imaging stages. Different events can be triggered in different imaging stages, such as changes in image intensity, changes in cell or organelle morphology, and protein or molecular movement. Furthermore, in the aforementioned phased imaging, high-throughput imaging (including large-field-of-view imaging or multi-point field-of-view imaging) or real-time 3D cell tracking can be actively or triggered based on cell motion characteristics, morphological changes, and number changes to ensure that images of the cell regions of interest and related regions are recorded during long-term live-cell imaging.

[0156] At different stages of the experiment, different imaging speeds and resolutions can be used based on the event's progression, cell movement speed, and changes in cell detail. For example, slow, low-resolution acquisition can reduce invalid data in stages where action is slow and specific cell details are not readily available; conversely, high-speed, high-resolution acquisition can be used in stages where action is rapid and specific cell action details need to be observed to obtain crucial data. Simultaneously, other fluorescence modalities can be triggered for imaging, reducing phototoxicity associated with prolonged fluorescence imaging. Throughout the entire real-time cell imaging process, intelligent decision-making imaging enables more efficient and higher-throughput microscopic imaging.

[0157] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this application. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this application. Such modifications, improvements, and corrections are suggested in this application, and therefore remain within the spirit and scope of the exemplary embodiments of this application.

[0158] Meanwhile, specific terms are used to describe embodiments of this utility model. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic related to at least one embodiment of this utility model. Therefore, it should be emphasized and noted that "an embodiment," "one embodiment," or "an alternative embodiment" mentioned twice or more in different positions in this utility model do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics in one or more embodiments of this utility model can be appropriately combined.

[0159] Finally, it should be understood that the embodiments described in this utility model are merely illustrative of the principles of the present utility model. Other modifications may also fall within the scope of this utility model. Therefore, alternative configurations of the embodiments of this utility model are considered to be consistent with the teachings of this utility model, rather than as examples or limitations. Accordingly, the embodiments of this utility model are not limited to those explicitly described and illustrated herein.

Claims

1. An automatic alignment system for ODT, comprising: The platform, the primary lifting platform, and the secondary lifting platform are characterized in that, The scanning illumination module and the secondary lifting platform are respectively installed on both sides of the platform along the Z-axis direction, and the platform is configured to support the scanning illumination module. The stage is slidably mounted on the primary lifting platform, which is configured to drive the scanning illumination module to move along the Z-axis. An objective lens holder is installed on the secondary lifting platform. The objective lens holder is used to install an illumination objective lens. The secondary lifting platform is configured to drive the illumination objective lens to move along the Z-axis.

2. The automatic alignment system according to claim 1, characterized in that, The primary lifting platform includes a first guide rail and a first motor. The first guide rail is arranged along the Z-axis direction, and the platform is slidably mounted on the first guide rail. The first motor drives the scanning illumination module to move along the Z-axis direction.

3. The automatic alignment system according to claim 2, characterized in that, The primary lifting platform includes a lead screw, which is connected to the first motor and the platform to convert the rotation of the first motor into linear displacement of the platform along the Z-axis.

4. The automatic alignment system according to claim 3, characterized in that, The lead screw is equipped with a backlash-eliminating nut.

5. The automatic alignment system according to claim 2, characterized in that, The primary lifting platform includes an encoder, which communicates with the first motor.

6. The automatic alignment system according to claim 1, characterized in that, The secondary lifting platform includes a second guide rail and a second motor. The second guide rail is arranged along the Z-axis direction. The objective lens holder is slidably mounted on the second guide rail. The second motor drives the illumination objective lens to move along the Z-axis direction.

7. The automatic alignment system according to claim 6, characterized in that, The secondary lifting platform includes a guide groove and a spring, the objective lens holder is connected to the spring, and the bearing of the second motor is installed in the guide groove.

8. The automatic alignment system according to claim 7, characterized in that, The guide groove is provided with a positioning groove.

9. The automatic alignment system according to claim 1, characterized in that, The automatic alignment system includes a target assembly configured to mount a target for automatic alignment, wherein the target assembly is mounted on the scanning illumination module on the side opposite to the stage along the Z-axis.

10. The automatic alignment system according to claim 9, characterized in that, The target assembly includes a substrate with a positioning hole on the substrate. The target is installed in the positioning hole and is located at the conjugate position in the illumination optical path of the scanning illumination module.

11. The automatic alignment system according to claim 10, characterized in that, The substrate is also provided with a vacancy hole, and the target assembly includes an adjustment member configured to adjust the position of the positioning hole and the vacancy hole.

12. The automatic alignment system according to claim 11, characterized in that, The adjusting component includes a handle and a slide groove. The handle is fixedly connected to the base, and the base is slidably connected to the slide groove.

13. The automatic alignment system according to claim 10, characterized in that, The target assembly also includes an adjustment knob for adjusting the position of the target.

14. The automatic alignment system according to claim 9, characterized in that, The target pattern is centered.

15. The automatic alignment system according to claim 1, characterized in that, The automatic alignment system includes a liquid level sensor mounted on the surface of the illumination objective, the liquid level sensor being configured to detect whether the illumination objective is in contact with a liquid surface.

16. The automatic alignment system according to claim 15, characterized in that, The liquid level sensor consists of multiple electrodes arranged in a cone shape.

17. The automatic alignment system according to claim 1, characterized in that, The automatic alignment system also includes a processor configured to determine whether the illumination objective is in contact with the liquid surface based on the brightness of the image captured by the camera.

18. The automatic alignment system according to claim 1, characterized in that, The automatic alignment system also includes a controller configured to control the movement of the primary lifting platform and / or the secondary lifting platform along the Z-axis.

19. A microscopic imaging apparatus, comprising an automatic alignment system, a scanning illumination module, and an imaging module as described in any one of claims 1 to 18, wherein the scanning illumination module illuminates a sample with rotating parallel light, which is transmitted through the sample and received by the imaging module, and the imaging module generates an image of the sample.

20. The microscopic imaging apparatus according to claim 19, characterized in that, It also includes a control module for controlling the scanning angle of the scanning galvanometer in the scanning illumination module, or controlling the number of illumination angles of the illumination module.

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