System and method for immersion objective bubble detection

Abstract

Systems and methods for efficiently detecting the presence or absence of a bubble in an immersion liquid between an immersion objective and a body are provided. For example, an automated imaging system includes an immersion objective, a light source configured to emit a first light toward a body via the immersion objective in a state where an immersion liquid is located on the immersion objective between the immersion objective and the body, wherein the body is configured to hold a sample, a first sensor configured to receive a first signal based on the first light after the first light reaches the body via the immersion objective, and a controller configured to detect the presence or absence of a bubble in the immersion liquid between the immersion objective and the body based on the first signal received by the first sensor.

Description

[0001] Cross-references to related applications This application is an international application claiming the benefit of U.S. Provisional Application No. 63 / 604,493, filed November 30, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] Embodiments of this disclosure relate to imaging systems and methods thereof, and more specifically to automated imaging systems and methods thereof capable of detecting air bubbles in a liquid between an immersion objective lens of an automated imaging system and a sample. Background Technology

[0003] Cell biology often uses immersion objectives (such as water) to increase light concentration and reduce changes in refractive index. These immersion objectives utilize the liquid between the objective and the sample. When the liquid between the objective and the sample contains air bubbles, the image may become blurry and degraded.

[0004] When air bubbles are present in the liquid between the objective lens and the sample, the focal length of the sample to be imaged will be greater than when there are no air bubbles in the liquid. This increased focal length also increases the risk of the sample coming into contact with laboratory glassware and damaging the objective lens.

[0005] In traditional manual microscopes, the operator needs to manually observe the application of the immersion liquid onto the immersion objective. If the operator visually observes air bubbles in the immersion liquid between the objective and the sample, they can manually adjust the operation by adding more liquid. This is cumbersome for the operator and slows down the operation. Furthermore, automated microscopes do not benefit from this method of manually guiding the liquid to the objective.

[0006] In automated imaging systems (such as microscopes), new instruments and methods are needed to efficiently detect the presence of air bubbles in the immersion liquid between the objective lens and the sample. Summary of the Invention

[0007] The embodiments disclosed herein can solve the above-mentioned problems and other issues.

[0008] According to embodiments of this disclosure, an automated imaging system can be provided, comprising an immersion objective, a light source, a first sensor, and a controller. The light source can be configured to emit first light toward the subject via the immersion objective when the immersion liquid is located on the immersion objective between the immersion objective and the subject. The subject is configured to hold a sample. The first sensor can be configured to receive a first signal based on the first light after the first light reaches the subject via the immersion objective. The controller can be configured to detect whether air bubbles exist in the immersion liquid between the immersion objective and the subject based on the first signal received by the first sensor.

[0009] According to embodiments of the present disclosure, the automatic imaging system may further include an immersion objective lens, wherein the light source may be further configured to emit a second light toward the subject via the immersion objective lens, the first sensor may be further configured to receive a second signal based on the second light after the second light reaches the subject via the immersion objective lens, and the controller may be further configured to detect whether there are air bubbles in the immersion liquid between the immersion objective lens and the subject based on the first signal and the second signal received by the first sensor.

[0010] According to embodiments of this disclosure, the controller may also be configured to detect the presence of air bubbles in the immersion liquid between the immersion objective and the body by comparing the ratio of the second signal to the first signal with a predetermined threshold.

[0011] According to embodiments of this disclosure, the controller may also be configured to detect the absence of air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being greater than a predetermined threshold, and to analyze the sample based on the detection that no air bubbles are present in the immersion liquid between the immersion objective and the body.

[0012] According to embodiments of this disclosure, the controller may also be configured to detect the presence of air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being less than or equal to a predetermined threshold.

[0013] According to embodiments of this disclosure, at least a portion of a first light emitted from a light source and at least a portion of a second light are reflected by a subject. A first sensor may be configured to receive a first signal based on the portion of the first light reflected by the subject and a second signal based on the portion of the second light reflected by the subject.

[0014] According to embodiments of this disclosure, the first light may be a first excitation light configured to cause the sample to emit a third light via fluorescence; the second light may be a second excitation light configured to cause the sample to emit a fourth light via fluorescence; and the first sensor may be configured to receive a first signal based on the third light emitted by the sample and to receive a second signal based on the fourth light emitted by the sample.

[0015] According to embodiments of this disclosure, the light source may be a narrow-beam light source including a laser diode.

[0016] According to embodiments of this disclosure, the first sensor may be an image sensor.

[0017] According to embodiments of this disclosure, the sensor may be adjacent to the light source, and the automated imaging system may include a second sensor, which is an image sensor that can be configured to image a sample.

[0018] According to embodiments of this disclosure, the light source may be a wide-beam light source including a lamp.

[0019] According to embodiments of the present disclosure, the automated imaging system may further include a rotating confocal microscope, which may include an immersion objective, a light source, a first sensor, a confocal turntable, at least one first optical body and at least one second optical body. The first optical body defines a first infinity correction optical region on a first side of the confocal turntable that is oriented toward the body holding the sample, and the second optical body defines a second infinity correction optical region on a second side of the confocal turntable that is oriented away from the body holding the sample.

[0020] According to embodiments of this disclosure, the light source and the first sensor are located in the first infinity-corrected optical region of the rotating confocal microscope.

[0021] According to embodiments of this disclosure, the light source and the first sensor are located in the second infinity-corrected optical region of the rotating confocal microscope.

[0022] According to embodiments of the present disclosure, the light source may be further configured to emit a second light toward the subject via the immersion objective when there is no immersion liquid on the immersion objective. The first sensor may be further configured to receive a second signal based on the second light after the second light reaches the subject via the immersion objective. The controller may be further configured to detect whether there are air bubbles in the immersion liquid between the immersion objective and the subject based on the first signal and the second signal received by the first sensor.

[0023] According to embodiments of this disclosure, a method performed by an automated imaging system may include: with an immersion liquid positioned on the immersion objective between the immersion objective and the main body of the automated imaging system, emitting a first light from a light source of the automated imaging system via the immersion objective toward the main body, wherein the main body may be configured to hold a sample; after the first light reaches the main body via the immersion objective, receiving a first signal based on the first light via a first sensor of the automated imaging system; and detecting the presence of air bubbles in the immersion liquid between the immersion objective and the main body based on the first signal received by the first sensor.

[0024] According to embodiments of this disclosure, the method performed by the automated imaging system may further include: emitting a second light toward the subject via a gas immersion objective of the automated imaging system through a light source; receiving a second signal based on the second light through a first sensor after the second light reaches the subject via the gas immersion objective; the detection may include detecting the presence of air bubbles in the immersion liquid between the liquid immersion objective and the subject based on the first signal and the second signal received by the first sensor.

[0025] According to embodiments of this disclosure, detection may include detecting the presence of air bubbles in the immersion liquid between the immersion objective and the body by comparing the ratio of the second signal to the first signal with a predetermined threshold.

[0026] According to embodiments of this disclosure, detection may include detecting the absence of air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being greater than a predetermined threshold, and the method may further include analyzing the sample based on the detection that no air bubbles are present in the immersion liquid between the immersion objective and the body.

[0027] According to embodiments of this disclosure, detection may include detecting the presence of air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being less than or equal to a predetermined threshold.

[0028] According to embodiments of this disclosure, the method may further include receiving at least a portion of a first light and at least a portion of a second light emitted from the light source by the subject reflecting the first light. Receiving a first signal may include receiving the first signal based on the portion of the first light reflected by the subject, and receiving a second signal may include receiving the second signal based on the portion of the second light reflected by the subject.

[0029] According to embodiments of this disclosure, emitting a first light causes the sample to emit a third light via fluorescence, emitting a second light causes the sample to emit a fourth light via fluorescence, receiving a first signal includes receiving a first signal based on the third light emitted by the sample, and receiving a second signal includes receiving a second signal based on the fourth light emitted by the sample.

[0030] According to embodiments of this disclosure, the method may further include emitting a second light from a light source to the subject via the immersion objective when there is no immersion liquid on the immersion objective; after the second light reaches the subject via the immersion objective, receiving a second signal based on the second light by a first sensor; detection may include detecting whether there are air bubbles in the immersion liquid between the immersion objective and the subject based on the first signal and the second signal received by the first sensor.

[0031] According to embodiments of this disclosure, a non-transitory computer-readable medium storing computer instructions can be provided. These computer instructions, when executed by at least one processor, cause the at least one processor to: With the immersion liquid positioned on the immersion objective between the immersion objective and the main body of the automatic imaging system, a first light is emitted from the light source of the automatic imaging system via the immersion objective to the main body, wherein the main body is configured to hold the sample; after the first light reaches the main body via the immersion objective, a first signal is received by a first sensor of the automatic imaging system based on the first light; and the presence of air bubbles in the immersion liquid between the immersion objective and the main body is detected based on the first signal received by the first sensor.

[0032] According to embodiments of this disclosure, the detection of air bubbles in the liquid between the objective lens and the sample can be automated and efficient.

[0033] According to embodiments of this disclosure, a sample can be imaged without human intervention to determine the presence of air bubbles in the liquid between the objective lens and the sample, thereby saving countless hours of error-prone manual labor.

[0034] Other aspects will be partially described in the following description, and some will become obvious from the description or can be learned by practicing the examples presented. Attached Figure Description

[0035] Further features, properties, and various advantages of the disclosed subject matter will become more apparent from the following detailed description and accompanying drawings, in which: Figure 1 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 2 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 3 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 4 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 5 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 6 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 7A This is a schematic diagram illustrating the detection of air bubbles in an immersion liquid according to an embodiment of the present disclosure; Figure 7B This is a schematic diagram illustrating that no air bubbles were detected in an immersion liquid according to an embodiment of the present disclosure; Figure 8A This is a flowchart illustrating an operation method of an automatic imaging system according to an embodiment of the present disclosure; Figure 8B This is a flowchart illustrating an operation method of an automatic imaging system according to an embodiment of the present disclosure; Figure 9 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 10 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 11 This is a schematic diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 12 This is a block diagram illustrating an automatic imaging system according to an embodiment of the present disclosure; Figure 13This is a schematic diagram illustrating a portion of an automated imaging system for implementing microplates according to embodiments of the present disclosure; and Figure 14 This is a schematic diagram illustrating a portion of an automated imaging system for a slide with a coverslip, according to an embodiment of the present disclosure. Detailed Implementation

[0036] Reference will now be made in detail to non-limiting exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. In the drawings, for clarity, the shape, size, etc., of certain components may be exaggerated or simplified.

[0037] In the following description, the same or similar reference numerals may be used for the same or similar parts in the accompanying drawings, and redundant descriptions of them may be omitted.

[0038] As will be understood by those skilled in the art, depending on the embodiments and functions, the term "lens" in this and throughout the description may refer to a single lens or a group of lenses.

[0039] According to embodiments of this disclosure, an automated imaging system is provided. The automated imaging system may include a microscope utilizing an immersion liquid (e.g., water) objective lens, and the automated imaging system may be configured to perform processes in which air bubbles can be detected within the immersion liquid (e.g., water) of the immersion objective lens.

[0040] For example, these processes may include multiple steps, including but not limited to: (1) emitting a first light from a light source toward a body holding the sample via an immersion objective; (2) recording a signal level (referred to as an air signal level) based on the intensity of the first light reflected from the body holding the sample; (3) replacing the immersion objective with a liquid immersion objective; (4) distributing an immersion liquid onto the liquid immersion objective; (5) emitting a second light from a light source toward the body holding the sample via the liquid immersion objective; (6) recording a signal level (referred to as a liquid (e.g., water) signal level) based on the intensity of the second light reflected from the body holding the sample; and (7) comparing the ratio of the air signal level to the liquid signal level with a predetermined threshold to determine whether the immersion liquid between the liquid immersion objective and the body contains air bubbles.

[0041] According to an embodiment, a low liquid signal level (e.g., low reflectivity of light emitted to the subject) can determine that there are no air bubbles in the immersion liquid between the immersion objective and the subject; a high liquid signal level (e.g., high reflectivity of light emitted to the subject) can determine that air bubbles are present in the immersion liquid between the immersion objective and the subject. The level of the reflected signal can be directly related to the refractive index change in the immersion liquid and air (e.g., air signal / liquid signal). For example, when air bubbles are present, the difference in refractive index between two measurements (e.g., air signal / liquid signal) can be greater than when no air bubbles are present.

[0042] The embodiments disclosed herein can implement the structure and function of the systems described in U.S. Patent No. 9,772,540B2 to Norris et al., U.S. Patent No. 10,072,982B2 to Zimenkov et al., and International Patent Application Publication No. WO2022120047A1 to Piette et al., the entire contents of which are incorporated herein by reference.

[0043] Figure 1 This is a schematic diagram illustrating an automatic imaging system according to a first embodiment of the present disclosure.

[0044] See Figure 1 The automated imaging system 3000 may include a first cube 3532, a mirror 3503, one or more immersion objectives 3522, a body 3523, a first lens 3524, a second lens 3525, a third lens 3526, a confocal turntable 3514, a second cube 3511, and a first sensor 3515. According to an embodiment, the automated imaging system 3000 may be configured as a turntable confocal microscope.

[0045] The first cube 3532 may be a structure (e.g., a body) including, for example, a light source 3510 and a dichroic mirror 3531. The light source 3510 may be configured to emit light 3501 toward the dichroic mirror 3531. As described in further detail below, the light source 3510 and its light 3501 can be used for bubble detection. The dichroic mirror 3531 may reflect the light 3501 toward a reflecting mirror 3503, which in turn may reflect the light 3501 toward an immersion objective 3522 toward the body 3523. That is, the light source 3510 may be configured to emit light 3501 toward the body 3523 via the immersion objective 3522. For example, the first cube 3532 may be used in a confocal mode relative to a confocal turntable 3514.

[0046] According to embodiments, light source 3510 can be, for example, a narrow-beam light source, such as, but not limited to, a laser diode (e.g., a solid-state laser or a semiconductor-based laser) and one or more light-emitting diodes (LEDs) capable of generating a narrow beam. For example, the narrow-beam light source in the embodiments of this disclosure, such as light source 3510, may include a laser diode with a wavelength of 635 nanometers to optimize camera efficiency and laser line availability. Increasing the laser wavelength of the laser diode can reduce phototoxicity, but may reduce camera efficiency. To address the problem of decreased camera efficiency, the laser power can be increased. Another advantage of using a longer wavelength is that the laser signal can be "guided" using a dichroic mirror without affecting the imaging optical path of the microscope, thereby enabling in-situ bubble detection.

[0047] Dichroic mirror 3531 can be partially reflective and partially transmissive. For example, some light can be reflected from the surface of dichroic mirror 3531, while other light can pass through the surface of dichroic mirror 3531.

[0048] One or more objectives 3522 may be configured to be located below the body 3523 holding the sample and to observe the sample from below. The one or more objectives 3522 may include, for example, one or more liquid immersion objectives and one or more gas immersion objectives.

[0049] Immersion objectives can be specially designed objectives to improve the resolution of a microscope. To immerse the objective 3522, which serves as an immersion objective, a drop of immersion liquid (such as water or other liquids that increase the refractive index, such as oil or glycerin) is placed on the objective 3522, and the liquid is held in place by its surface tension. The objective 3522 can then be brought to the sample, trapping the droplet between the sample and the objective 3522. In this way, light traveling between the sample and the objective 3522 will not pass through air. The higher refractive index of the liquid relative to air results in an increased numerical aperture of the objective 3522, providing stronger light-gathering ability. Therefore, the increase in numerical aperture improves resolution and enhances the signal level when imaging the sample. Furthermore, as described below with reference to Equation 1, a fluid with a higher refractive index (relative to air) reduces reflectivity because the closer the refractive index, the less reflection occurs when transferring from one substance to another.

[0050] According to an embodiment, the objective lens 3522 (i.e., the liquid immersion objective lens) can be brought to the sample, and then (manually or automatically) a droplet can be placed on the objective lens 3522.

[0051] An immersion objective (also known as a dry objective) is designed to work without any immersion liquid. That is, when using objective 3522 as an immersion objective, air (instead of an immersion liquid) can be trapped between the sample and objective 3522.

[0052] One or more objective lenses 3522 may be moved via a moving mechanism 3530. The moving mechanism 3530 may include, for example, at least one actuator. According to an embodiment, the moving mechanism 3530 may be configured to move one or more objective lenses 3522 along at least one of a vertical, horizontal, and / or rotational direction to exchange the objective lenses 3522 to an observation position below the body 3523, for example, for imaging a sample held by the body 3523. Furthermore, the moving mechanism 3530 may be configured to move one or more of the objective lenses 3522 in the observation position in the vertical direction to change the sharpness of the image obtained using the one or more objective lenses 3522. According to an embodiment, the moving mechanism 3530 may include an objective lens stage for holding the objective lenses 3522 and may be configured to rotate under the control of the actuator of the moving mechanism 3530, thereby enabling the objective lenses 3522 to be selectively positioned in the observation position.

[0053] The body 3523 can be configured to hold the sample in a fixed position within the automated imaging system 3000, thereby enabling imaging using the objective lens 3522. According to an embodiment, the body 3523 can be, for example, a microplate 310 (see...). Figure 13 ) or coverslip 320 (see Figure 14 ).

[0054] See Figure 13 The microplate 310 can hold the sample S within the wells 316 of the microplate 310. The microplate 310 may include a base plate 312 and sidewalls 314 defining one or more wells 316. The base plate 312 and / or sidewalls 314 may be made of a transparent material. For example, the base plate 312 and / or sidewalls 314 may be made of glass or plastic.

[0055] Objective lens 3522 can be located below microplate 310. When objective lens 3522 is a liquid immersion objective, an immersion liquid L (e.g., water) can be provided between objective lens 3522 and microplate 310. For example, the immersion liquid L can be in direct contact with the lens of objective lens 3522 and the bottom surface of base plate 312 of microplate 310. When objective lens 3522 is a gas immersion objective, air can be provided between objective lens 3522 and microplate 310 instead of an immersion liquid. For example, air can be in direct contact with the lens of objective lens 3522 and the bottom surface of base plate 312 of microplate 310.

[0056] According to an embodiment, when a sample S is provided, the input light 301 of the automated imaging system 3000 can pass upward through the objective lens 3522, the immersion liquid L, or air, and then through the base plate 312 of the microporous plate 310 to reach the sample S in the aperture 316. In response, the output light 302 can be provided from the sample S and can pass downward through the base plate 312, the immersion liquid L, or air, and then through the objective lens 3522, thereby being received by a sensor (e.g., the first sensor 3515) of the automated imaging system (e.g., the automated imaging system 3000). For example, the output light 302 can be a portion of the input light 301 reflected from the sample S, or it can be light emitted by the fluorescence of the sample S in response to the receipt of the input light 301. According to an embodiment, the input light 301 can be light output from any light source of the automated imaging system (e.g., the automated imaging system 3000) in the embodiments of this disclosure. For example, the input light may be light 3501 emitted by light source 3510 and / or light emitted by light source 3512 of second cube 3511, but the embodiments of this disclosure are not limited thereto.

[0057] According to an embodiment, when performing bubble detection (explained further below), sample S can be omitted, and at least a portion of the input light 301 (e.g., Figure 1 The light 3501 (e.g., from the input light 301) can be reflected by the base plate 312 of the microporous plate 310. That is, the output light 302 can be the portion of the input light 301 reflected by the base plate 312. For example, the output light 302 can be... Figure 1 The reflected light 3502 is shown. Then, the automatic imaging system in this embodiment of the present disclosure can use the output light 302 to determine whether there are air bubbles in the immersion liquid L.

[0058] According to an embodiment, bubble detection can be performed in the presence of a sample S. For example, at least a portion of the input light 301 may be reflected by the sample S, or the sample S may emit fluorescence based on the input light 301 as output light 302, which can be used by the automated imaging system of this disclosure embodiment to determine whether bubbles are present in the immersion liquid L.

[0059] See Figure 14 The sample S can be placed on a glass slide 330 (such as a microscope slide), and a coverslip 320 can be placed on the glass slide 330, so that the glass slide 330 and the coverslip 320 hold the sample S between them. The glass slide 330 and / or the coverslip 320 can be made of a transparent material. For example, the glass slide 330 and / or the coverslip 320 can be made of glass or plastic.

[0060] According to an embodiment, when a sample S is provided, the input light 301 of the automated imaging system 3000 can pass upward through the objective lens 3522, the immersion liquid L, or air, and then through the slide 330 to reach the sample S. In response, the output light 302 can be provided from the sample S and can pass downward through the slide 330, the immersion liquid L, or air, and then through the objective lens 3522, thereby being received by a sensor (e.g., the first sensor 3515) of the automated imaging system (e.g., the automated imaging system 3000). As described above, the output light 302 can be a portion of the input light 301 reflected from the sample S, or it can be light emitted by the fluorescence of the sample S in response to the receipt of the input light 301. As described above, the input light 301 can be light output from any light source of the automated imaging system (e.g., the automated imaging system 3000) in this disclosure embodiment. For example, the input light can be light 3501 emitted by the light source 3510 and / or light emitted by the light source 3512 of the second cube 3511.

[0061] According to an embodiment, when performing bubble detection (explained further below), sample S can be omitted, allowing coverslip 320 to directly contact slide 330. In this case, input light 301 (e.g., Figure 1 The input light 301 can reach the coverslip 320 through the slide 330. At least a portion of the input light 301 can be reflected by the coverslip 320. That is, the output light 302 can be the portion of the input light 301 reflected by the coverslip 320. For example, the output light 302 can be... Figure 1 The reflected light 3502 is shown. Then, the automatic imaging system in this embodiment of the present disclosure can use the output light 302 to determine whether there are air bubbles in the immersion liquid L.

[0062] According to an embodiment, bubble detection can be performed in the presence of a sample S. For example, at least a portion of the input light 301 may be reflected by the sample S, or the sample S may emit fluorescence based on the input light 301 as output light 302, which can be used by the automated imaging system of this disclosure embodiment to determine whether bubbles are present in the immersion liquid L.

[0063] According to an embodiment, the sensing signal level of the output light 302 can depend on whether the input light 301 is reflected by the body 3523 (or the sample S) or emitted by the sample S via fluorescence. For example, when reflected light (such as light from a narrow beam) is sensed, the sensed liquid signal may be lower than the signal when bubbles are present in the immersion liquid if no bubbles are present. In contrast, when light emitted by the sample via fluorescence is sensed, the sensed liquid signal may be higher than the signal when bubbles are present in the immersion liquid if no bubbles are present.

[0064] Further reference Figure 1After the light 3501 emitted by the light source 3510 reaches the body 3523, a portion of the light 3501 can be reflected by the body 3523, becoming reflected light 3502. The reflected light 3502 can pass downward through the immersion objective lens 3522 so that it can be received by the first sensor 3515. For example, the reflected light 3502 can be reflected by the mirror 3503 and pass sequentially through the dichroic mirror 3531, the first lens 3524, the confocal turntable 3514, the second lens 3525, the dichroic mirror 3516 of the second cube 3511, and then through the third lens 3526 before being received by the first sensor 3515.

[0065] According to an embodiment, the first lens 3524, the second lens 3525, and / or the third lens 3526 may be tubular lenses. The rear portions of the first lens 3524 and the immersion objective 3522 may define a first infinity correction optical region 3504 therebetween, while the second lens 3525 and the third lens 3526 may define a second infinity correction optical region 3505 therebetween. That is, the first infinity correction optical region 3504 may be located on one side of the confocal turntable 3514 facing the body 3523 holding the sample, while the second infinity correction optical region 3505 may be located on the other side of the confocal turntable 3514 facing the first sensor 3515. The infinity correction optical region may be a region that provides parallel light rays (i.e., light rays intersect at infinity).

[0066] The confocal turntable 3514 can be configured to provide an optical path (e.g., an excitation path, a reflection path, and / or an emission path) for the automated imaging system 3000. For example, light from the sample or subject 3523 (such as reflected light 3502) and light from a light source (such as light source 3512) can pass through the confocal turntable 3514. According to embodiments, the confocal turntable 3514 may include a pattern of pinholes or slits providing the optical path. These pinholes or slits may be spaced far apart to act optically independently and may be arranged in multiple spirals along the confocal turntable 3514. In an exemplary embodiment, the confocal turntable 3514 may be approximately 2 mm thick and made of glass or quartz. The confocal turntable 3514 may be coated to be opaque or have a given transparency or opacity, except leaving transparent areas as pinhole or slit patterns. The surface of the confocal turntable 3514 may be configured not to reflect oncoming light. According to an embodiment, the confocal turntable 3514 can be configured to rotate based on the drive of at least one actuator in the automated imaging system 3000. According to an embodiment, the confocal turntable 3514 can be controlled to rotate continuously, thereby scanning the sample. As the confocal turntable 3514 rotates, the sample can be illuminated one point at a time, and a complete sample image can be detected on a sensor (such as a first sensor 3515) to reconstruct a complete image of the sample. According to an embodiment, the confocal turntable 3514 can employ the turntable configuration described in International Patent Application Publication No. WO2022120047A1 by Piette et al.

[0067] The second cube 3511 may be a structure (e.g., a body) including, for example, a light source 3512, a lens 3513 (e.g., an emission filter), and a dichroic mirror 3516. The light source 3512 may be configured to emit light to the dichroic mirror 3516 via the lens 3513. The lens 3513 may be configured to block light of a specific wavelength from the light source 3512 while allowing other light of other specific wavelengths from the light source 3512 to pass through. For example, the lens 3513 may be configured to form a bandpass for exciting a sample held by the body 3523. The dichroic mirror 3516 may be partially reflective and partially transmissive. For example, some light may be reflected from the surface of the dichroic mirror 3516, while other light may pass through the surface of the dichroic mirror 3516.

[0068] According to an embodiment, the light source 3512 can be configured to emit light toward a sample held by the body 3523 so as to image the sample using the light. For example, the light emitted by the light source 3512 can pass through lens 3513, be reflected from dichroic mirror 3516, pass through second lens 3525, confocal turntable 3514, first lens 3524 and dichroic mirror 3531, be reflected from mirror 3503, and then pass through immersion objective lens 3522 (as...). Figures 13-14 The input light 301 reaches the sample. Based on the light from the light source 3512, the output light ( Figures 13-14 The output light (302) can be reflected from or generated by the sample (e.g., by fluorescence), and can pass downward through the immersion objective 3522 to be received by the first sensor 3515 to acquire an image of the sample for analysis. For example, the output light can be reflected by mirror 3503 and pass through dichroic mirror 3531, first lens 3524, confocal turntable 3514, second lens 3525, second cube 3511, and dichroic mirror 3516, then through third lens 3526, before being received by the first sensor 3515. According to an embodiment, the light source 3512 can be a confocal excitation source. The confocal excitation source can be any light source suitable for confocal microscopy. For example, the light source 3512 can be a solid-state light source (e.g., one or more LEDs) or a solid-state laser or a semiconductor-based laser (e.g., a laser diode).

[0069] The first sensor 3515 may be configured to receive light in response to light emitted by the light source 3510 and / or the light source 3512 toward the subject 3523. For example, the first sensor 3515 may be configured to acquire an image of the sample by receiving reflected light caused by light emitted by the light source (such as the light source 3512) toward the subject 3523 or light generated by the sample (such as by fluorescence), as a controller 70 of the automated imaging system 3000 (see [link to controller 70]). Figure 12 As part of the analysis, the first sensor 3515 (or another sensor) may be further configured to acquire a signal by receiving reflected light 3502, which is a reflected portion of the light 3501 emitted by the light source 3510, to detect the presence of air bubbles in the immersion liquid between the immersion objective 3522 (i.e., the immersion objective) and the body 3523.

[0070] The first sensor 3515 can be, for example, an imaging camera, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) detector, a line sensor, or a single-crystal photodiode. For example, the imaging camera can have more than one million pixels, with each pixel having a size of 3-6 µm.

[0071] The controller 70 of the automatic imaging system 3000 (see...) Figure 12The system can be configured to detect the presence of air bubbles in the immersion liquid between the immersion objective 3522 (i.e., the immersion lens) and the main body 3523 based on the reflected light 3502 received by the first sensor 3515 when the automatic imaging system 3000 is in two different states. The first state (hereinafter referred to as the dry state) can be when there is no immersion liquid on the immersion objective 3522 through which the light 3501 and reflected light 3502 pass. The second state (hereinafter referred to as the wet state) can be when there is indeed immersion liquid on the same or different immersion objectives 3522 through which the light 3501 and reflected light 3502 pass. For the dry state, the immersion objective 3522 used can be a gas-immersion objective or an immersion objective that is not currently coated with immersion liquid. For the wet state, the immersion objective 3522 used can be an immersion objective with immersion liquid on it, and this objective can be different from the gas-immersion or immersion objective used in the dry state, or it can be the same immersion objective used in the dry state. In a dry state, the reflected light 3502 obtained by the first sensor 3515 (or another sensor) can be referred to as an air signal. In a wet state, the reflected light 3502 obtained by the first sensor 3515 (or another sensor) can be referred to as a liquid signal.

[0072] According to an embodiment, the controller 70 can acquire air and liquid signals from the first sensor 3515 and record the air and liquid signals. The controller 70 can be configured to compare the ratio of the air signal to the liquid signal with a predetermined value (e.g., a predetermined threshold) to detect the presence of air bubbles in the immersion liquid between the immersion objective 3522 and the body 3523 in a wet state. Details of how the controller 70 can detect the presence of air bubbles according to embodiments of this disclosure are provided below.

[0073] During the wet state, the intensity of the liquid signal (e.g., reflected light 3502) varies based on the presence or absence of air bubbles in the immersion liquid on the immersion objective 3522. For example, the percentage of reflectance of light 3501 from the body 3523 is affected by the presence or absence of air bubbles. High reflectance can indicate a significant change in refractive index, thus indicating the application of a poor-quality immersion fluid containing air bubbles. Low reflectance can indicate a uniform and high-quality application of the immersion fluid on the immersion objective 3522 (e.g., the absence of air bubbles).

[0074] For example, refer to Figure 7A When the immersion liquid 1001 on the immersion objective lens includes air bubbles, it is configured to hold the sample body 3523 (reference). Figure 1 This will produce a strong reflection R1. In contrast, the reference... Figure 7B When the immersion liquid 1002 on the immersion objective does not contain any air bubbles, it is configured to hold the sample body 3523 (reference). Figure 1 This will produce a weak reflection R2.

[0075] The expected reflectivity can be calculated using Fresnel's formula, which simplifies the normal incidence, as shown in formula (1) below.

[0076] (Formula 1) In Formula 1, n t It is the refractive index of the medium (e.g., liquid or air) between the immersion objective 3522 and the sample-holding body 3523, n i R is the refractive index of the main body 3523, while R is the reflectivity of light 3501.

[0077] For example, when the medium is water, n t It can be 1.333 when the medium is air (e.g., no immersion fluid on an immersion objective), n t It can be 1, when the main body 3523 is glass, n i It can be 1.5. In the first case (e.g., in a wet state), the medium is water in which there are no air bubbles (n t =1.333) and the main body 3523 is glass (n t =1.5), the expected reflectivity (R) could be 0.35%. In the second case (e.g., in a dry state), where the medium is air (n t =1), while the main body 3523 is glass (n t =1.5), the expected reflectance (R) can be 4%. Therefore, the reflectance of air to water medium (4 / 0.35) is 11.5.

[0078] In a wet state, if the immersion liquid (e.g., water) is properly positioned on the immersion objective 3522 without any air bubbles, the intensity of the liquid signal received by the controller 70 is expected to be approximately 11.5 times weaker than the intensity of the air signal received by the controller 70 in a dry state. In contrast, if air bubbles are present in the immersion fluid on the immersion objective 3522 in a wet state, the intensity of the liquid signal received by the controller 70 is expected to be closer to the intensity of the air signal received by the controller 70 in a dry state. For example, when air bubbles are present in a wet state, the ratio of the air signal to the liquid signal can be much less than 11.5, approximately 1.

[0079] In view of the above, for a wet state, the controller 70 can determine whether air bubbles exist in the immersion liquid on the immersion objective lens 3522 based on the ratio of the air signal to the liquid signal. For example, the controller 70 can compare the ratio of the air signal to the liquid signal with a predetermined threshold to make the determination.

[0080] According to an embodiment, the controller 70 can determine (or detect) the absence of air bubbles in the immersion liquid between the immersion objective 3522 (immersion objective) and the body 3523 based on the ratio of the air signal to the liquid signal being greater than a predetermined threshold. Then, based on the detection that no air bubbles are present on the immersion objective 3522, the controller 70 can automatically cause the automated imaging system 3000 to perform analysis, including imaging the sample (e.g., using the light source 3512 and the immersion objective 3522).

[0081] According to an embodiment, controller 70 can determine (or detect) the presence of air bubbles in the immersion liquid between immersion objective 3522 (immersion liquid objective) and body 3523 based on the ratio of air signal to liquid signal being less than or equal to a predetermined threshold. Based on the detection of air bubbles, controller 70 may not automatically perform analysis (e.g., imaging of the sample). For example, controller 70 may enable output device 20 (see...) Figure 12 The system outputs an error alert to the user so that the user can manually check for air bubbles in the immersion fluid between the immersion objective and the body 3523. If the user confirms the presence of air bubbles, the user can manually reapply the immersion fluid to the immersion objective 3522, or via input device 10 (see [link]). Figure 12 The input on the device instructs controller 70 to automatically reapply the immersion liquid to immersion objective 3522. Afterward, controller 70 can re-perform bubble detection. Alternatively, if the user confirms that no bubbles are present, the user can input the bubble via input device 10 (see [link]). Figure 12 The input on the device is used to cover errors, causing the controller 70 to perform analysis (e.g., capturing sample images using the light source 3512 and the immersion objective 3522).

[0082] According to embodiments of this disclosure, the predetermined threshold can be determined or obtained in various ways (e.g., by the controller 70). For example, the controller 70 can determine the predetermined threshold based on its image analysis results and / or bubble detection results, or the controller 70 can do so through, for example, a user using input device 10 (see...). Figure 12 Input a predetermined threshold into the controller 70 to obtain the predetermined threshold.

[0083] Figure 2 This is a schematic diagram illustrating an automatic imaging system 3000A according to a second embodiment of the present disclosure.

[0084] Except for the second sensor 3521, which can be located within the first cube 3532 and adjacent to the light source 3510, the automatic imaging system 3000A can be... Figure 1 The automatic imaging system 3000 shown is the same as or similar to that shown. For example, the second sensor 3521 may be located in the first infinity correction optical region 3504.

[0085] According to a second embodiment, instead of using the first sensor 3515 for bubble detection as described above, a second sensor 3521 can be used for bubble detection. For example, the second sensor 3521 can be configured for both air and liquid signals (e.g., reflected light 3502 in dry and wet conditions) for bubble detection. For example, reflected light 3502 can be received by the second sensor 3521 by being reflected by a reflector 3503 and a dichroic mirror 3531. Therefore, the controller 70 can obtain air and liquid signals for bubble detection from the second sensor 3521 and perform sample analysis, including image imaging of the sample, using only the first sensor 3515. According to an embodiment, the second sensor 3521 can be a dedicated sensor for bubble detection. The second sensor 3521 can have the same or different configuration as the first sensor 3515 and can be, for example, an imaging camera, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) detector, a line sensor, or a single-crystal photodiode. By including a second sensor 3521 co-located with a corresponding light source 3510, faster and simpler bubble detection can be provided.

[0086] According to an embodiment, in addition to bubble detection, the controller 70 can also be configured to perform autofocusing of sample imaging as described by Norris et al. For example, the first cube 3532 may have the configuration of the autofocusing module of Norris et al., and the light source (e.g., laser) and sensor in such autofocusing module may be controlled by the controller 70 to perform bubble detection and autofocusing.

[0087] Figure 3 This is a schematic diagram illustrating an automatic imaging system 3000B according to a third embodiment of the present disclosure.

[0088] In addition to the first cube 3532 being replaced by the first cube 3532A, the automatic imaging system 3000B can be used with... Figure 1 The automatic imaging system shown is the same as or similar to the 3000.

[0089] For example, although Figure 1 The first cube 3532 includes a light source 3510, which may be a narrow beam light source, but Figure 3The first cube 3522A includes a light source 3510A, which may be a wide-beam light source. The wide-beam light source may be, for example, a lamp (e.g., a light-emitting diode (LED), a halogen tungsten arc lamp, a mercury arc lamp, a xenon arc lamp, a laser diode, etc.). For example, the first cube 3532A can be used for imager of a sample in a wide-field mode. Furthermore, a lens 3541A may be located within the first cube 3532A. The lens 3541A may be configured to collimate light 3501 onto the back surface of the immersion objective 3522. Similar to the description of the light source 3510 in the first embodiment, the light source 3510A and its light 3501 can be used for bubble detection.

[0090] Figure 4 This is a schematic diagram illustrating an automatic imaging system 3000C according to a fourth embodiment of the present disclosure.

[0091] Except that the first cube 3532 is located between the second lens 3525 and the third lens 3526, within the second infinity correction optical region 3505, and not outside the first infinity correction optical region 3504, the automatic imaging system 3000C can be with Figure 1 The automatic imaging system 3000 shown is the same as or similar to that shown. Furthermore, a third cube 3517 may be disposed within the first infinity correction optical region 3504, between the reflector 3503 and the first lens 3524. Figure 1 and Figure 4 The functions of the first cubes 3532 in the process can be similar to each other. That is, for example, light 3501 from light source 3510 can still be used to perform bubble detection, as described in the embodiments of this disclosure.

[0092] The third cube 3517 may be a structure (e.g., a main body) including, for example, a light source 3518, a lens 3520 (e.g., a filter), and a dichroic mirror 3519. According to embodiments of this disclosure, the third cube 3517 may be similar to the second cube 3511. However, according to embodiments, the light source 3512 of the second cube 3511 may be a wide-beam laser light source for wide-angle imaging, positioned to utilize confocal optics (e.g., a confocal turntable 3514), while the light source 3518 of the third cube 3517 may be a wide-beam LED light source, not positioned to utilize confocal optics (e.g., a confocal turntable 3514).

[0093] Light source 3518 can be configured to emit light toward dichroic mirror 3519 via lens 3520. According to an embodiment, light source 3518 can be a confocal excitation source. A confocal excitation source can be any light source suitable for confocal microscopy. For example, light source 3518 can be a solid-state light source (e.g., one or more LEDs) or a solid-state laser or a semiconductor-based laser (e.g., a laser diode).

[0094] Lens 3520 can be configured to block light of a specific wavelength from light source 3518 while allowing other light of other specific wavelengths from light source 3518 to pass through. For example, lens 3520 can be configured to form a bandpass for exciting a sample held by body 3523. Dichroic mirror 3519 can be partially reflective and partially transmissive. For example, some light may be reflected from the surface of dichroic mirror 3519, while other light may pass through the surface of dichroic mirror 3519. According to an embodiment, dichroic mirror 3519 may reflect light toward mirror 3503, which may reflect light toward immersion objective 3522 toward body 3523. That is, light source 3518 can be configured to emit light toward the body via immersion objective 3522. According to an embodiment, first sensor 3515 can be configured to perform sample analysis (e.g., sample imaging) based on receiving light reflected or generated by the sample based on light from light source 3518.

[0095] According to an embodiment, the automated imaging system 3000C may include at least one actuator configured to move one or both of the first cube 3532 and the second cube 3511 into and out of a position between the second lens 3525 and the third lens 3526. For example, at least one actuator (based on control of the controller 70) may be configured to exchange the first cube 3532 and the second cube 3511 for a position between the second lens 3525 and the third lens 3526, such that the automated imaging system 3000C can selectively use the first cube 3532 for bubble detection or the second cube 3511 for sample analysis. Alternatively, at least one actuator (based on control of the controller 70) may be configured to move one or both of the first cube 3532 and the second cube 3511 such that the first cube 3532 and the second cube 3511 are coaxially positioned one after the other between the second lens 3525 and the third lens 3526. Alternatively, the first cube 3532 and the second cube 3511 can be coaxially fixed one after the other between the second lens 3525 and the third lens 3526, with some transmissive optical elements in between.

[0096] According to an embodiment, the controller 70 can be configured to selectively perform sample analysis using a sample image obtained by the first sensor 3515 based on the light emitted by the light source 3512 of the second cube 3511 and / or the light emitted by the light source 3518 of the third cube 3517.

[0097] Figure 5 This is a schematic diagram illustrating an automatic imaging system 3000D according to a fifth embodiment of the present disclosure.

[0098] Besides the second sensor 3521 being located within the first cube 3532, adjacent to the light source 3510, the automatic imaging system 3000D can be... Figure 4 The automatic imaging system 3000C shown is the same as or similar to that described. For example, the second sensor 3521 may be located in the second infinity correction optical region 3505.

[0099] According to the fifth embodiment, compared with the second embodiment (see...), Figure 2 Similarly, bubble detection can be performed using a second sensor 3521 instead of the first sensor 3515 as described above. For example, the second sensor 3521 can be configured to receive reflected light 3502 constituting air and liquid signals for bubble detection. For example, the reflected light 3502 can be received by the second sensor 3521 by reflection from mirror 3503, passing through dichroic mirror 3519, first lens 3524, and confocal turntable 3514, and then reflected from dichroic mirror 3531. Therefore, the controller 70 can obtain air and liquid signals for bubble detection from the second sensor 3521 and perform sample analysis, including image imaging of the sample, using only the first sensor 3515. According to embodiments, the second sensor 3521 can be a dedicated sensor for bubble detection. The second sensor 3521 can have the same or different configuration as the first sensor 3515 and can be, for example, an imaging camera, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS)-based detector, a line sensor, or a single-crystal photodiode. By including a second sensor 3521 co-located with the corresponding light source 3510, faster and simpler bubble detection can be provided.

[0100] According to an embodiment, in addition to bubble detection, the controller 70 can also be configured to perform autofocusing of sample imaging as described by Norris et al. For example, the first cube 3532 may have the configuration of the autofocusing module of Norris et al., and the light source (e.g., laser) and sensor in such autofocusing module may be controlled by the controller 70 to perform bubble detection and autofocusing.

[0101] According to the embodiment, the light source 3510 within the first cube 3532, i.e., the narrow-beam light source, can be light source 3510A within the first cube 3532 (see [reference]). Figure 3 That is, a wide-beam light source is used instead, similar to the description above regarding the third embodiment (see...). Figure 3 ).

[0102] Figure 6 This is a schematic diagram illustrating an automatic imaging system 3000E according to a sixth embodiment of the present disclosure.

[0103] Except for omitting the second cube 3511 and replacing the first cube 3532 with the first cube 3532B within the second infinity correction optical region 3505, the automatic imaging system 3000E can be used with... Figure 4 The automatic imaging system 3000C shown is the same as or similar to the one described.

[0104] For example, although Figure 4 The first cube 3532 includes a light source 3510 as a narrow beam light source, but Figure 6 The first cube 3532B includes a light source 3510B as a wide-beam light source. The wide-beam light source can be, for example, a lamp (e.g., an LED, a halogen tungsten arc lamp, a mercury arc lamp, a xenon arc lamp, a laser diode, etc.). Furthermore, a lens 3541B can be located within the first cube 3532B to focus the wide-beam light source to infinity. (See also: [link to first embodiment]). Figure 1 Similar to the above, the light source 3510B and its light 3501 can be used for bubble detection.

[0105] Figure 9 This is a schematic diagram illustrating an automatic imaging system 3000F according to a seventh embodiment of the present disclosure.

[0106] Besides omitting the confocal turntable 3514, the second lens 3525, the second cube 3511, and the third lens 3526, the automatic imaging system 3000F can be integrated with... Figure 1 The automated imaging system 3000 shown is the same as or similar to that shown. For example, the automated imaging system 3000F can be configured as a digital microscope instead of a rotating confocal microscope, wherein the first cube 3532 is positioned in the infinity correction optical region of the digital microscope.

[0107] In addition, such as Figure 9 As shown, a second cube 3511A can be provided to replace the second cube 3511 (see...). Figure 1 ).

[0108] The second cube 3511A may be a structure (e.g., a body) including, for example, a light source 3512A, a lens 3513A (e.g., an emission filter), and a dichroic mirror 3516A. The light source 3512A may be configured to emit light toward the dichroic mirror 3516A via the lens 3513A. The lens 3513A may be configured to block light of a specific wavelength from the light source 3512A while allowing other light of other specific wavelengths from the light source 3512A to pass through. For example, the lens 3513A may be configured to form a bandpass for exciting a sample held by the body 3523. The dichroic mirror 3516A may be partially reflective and partially transmissive. For example, some light may be reflected from the surface of the dichroic mirror 3516A, while other light may pass through the surface of the dichroic mirror 3516A.

[0109] According to an embodiment, the light source 3512A can be configured to emit light toward a sample held by the body 3523 so as to image the sample using the light. For example, the light from the light source 3512A can pass through the lens 3513A, be reflected from the dichroic mirror 3516A, be reflected from the mirror 3503, and then pass through the immersion objective 3522 to reach the sample (e.g., as a liquid-immersed objective lens). Figures 13-14 Input light 301 in the light source 3512; output light (e.g., based on the light from the light source 3512). Figures 13-14 The output light (302) can be reflected from or generated by the sample (e.g., by fluorescence), and can pass downward through the immersion objective 3522 to be received by the first sensor 3515, thereby obtaining an image of the sample. For example, the output light can be reflected by the mirror 3503 and passed through the dichroic mirror 3516A and the first lens 3524 before being received by the first sensor 3515. According to an embodiment, the light source 3512A can be a confocal excitation source. The confocal excitation source can be any light source suitable for confocal microscopy. For example, the light source 3512A can be a solid-state light source (e.g., one or more LEDs) or a solid-state laser or a semiconductor-based laser (e.g., a laser diode). According to an embodiment, the light source 3512A can be a wide-field fluorescence excitation source (e.g., a lamp).

[0110] According to an embodiment, the automated imaging system 3000F may include at least one actuator configured to move one or both of the first cube 3532 and the second cube 3511A to and out of a position between the reflector 3503 and the first lens 3524. For example, at least one actuator (based on control of the controller 70) may be configured to exchange the first cube 3532 and the second cube 3511A for a position between the reflector 3503 and the first lens 3524, such that the automated imaging system 3000F can selectively use the first cube 3532 for bubble detection or the second cube 3511A for sample analysis. Alternatively, at least one actuator (based on control of the controller 70) may be configured to move one or both of the first cube 3532 and the second cube 3511A such that the first cube 3532 and the second cube 3511A are coaxially positioned one after the other between the reflector 3503 and the first lens 3524. Alternatively, the first cube 3532 and the second cube 3511A can be coaxially fixed one after the other between the reflector 3503 and the first lens 3524, with some transmissive optical elements in between.

[0111] Figure 10 This is a schematic diagram illustrating an automatic imaging system 3000G according to an eighth embodiment of the present disclosure.

[0112] Besides the second sensor 3521 being located within the first cube 3532, adjacent to the light source 3510, the automatic imaging system 3000G can be... Figure 9 The automatic imaging system shown is the same as or similar to the 3000F, and is similar to... Figure 2 The second embodiment is shown.

[0113] According to the eighth embodiment, instead of using the first sensor 3515 for bubble detection as described above, a second sensor 3521 can be used for bubble detection. For example, the second sensor 3521 can be configured to receive air and liquid signals (e.g., reflected light 3502 in dry and wet states) to detect bubbles. For example, reflected light 3502 can be received by the second sensor 3521 by being reflected by a reflector 3503 and a dichroic mirror 3531. Therefore, the controller 70 can obtain air and liquid signals for bubble detection from the second sensor 3521 and use the first sensor 3515 alone for sample analysis, including imaging the sample. According to the embodiment, the second sensor 3521 can be a dedicated sensor for bubble detection. The second sensor 3521 can have the same or different configuration as the first sensor 3515 and can be, for example, an imaging camera, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS)-based detector, a line sensor, or a single-crystal photodiode. By including a second sensor 3521 co-located with the corresponding light source 3510, faster and simpler bubble detection can be provided.

[0114] According to an embodiment, in addition to bubble detection, the controller 70 can also be configured to perform autofocusing of sample imaging as described by Norris et al. For example, the first cube 3532 may have the configuration of the autofocusing module of Norris et al., and the light source (e.g., laser) and sensor in such autofocusing module may be controlled by the controller 70 to perform both bubble detection and autofocusing.

[0115] Figure 11 This is a schematic diagram illustrating an automatic imaging system 3000H according to a ninth embodiment of the present disclosure.

[0116] Except for the first cube 3532, which was Figure 3 Besides replacing the first cube 3532A, the automatic imaging system 3000H can be used with Figure 9 The automatic imaging system shown is the same as or similar to the 3000F.

[0117] For example, although Figure 9 The first cube 3532 includes a light source 3510 as a narrow beam light source, but Figure 3The first cube 3522A includes a power supply 3510A as a wide-beam light source. The wide-beam light source can be, for example, a lamp (e.g., an LED, a halogen tungsten arc lamp, a mercury arc lamp, a xenon arc lamp, a laser diode, etc.). Furthermore, a lens 3541A can be located within the first cube 3532A. The lens 3541A can be configured to collimate light 3501 to the back surface of the immersion objective lens 3522. (See also the light source 3510 of the first embodiment). Figure 1 Similar to the above, the light source 3510A and its light 3501 can be used for bubble detection.

[0118] As described above, according to embodiments of the present disclosure, the controller 70 can be configured to determine, in a wet state, whether air bubbles exist in the immersion liquid between the immersion objective lens 3522 and the body 3523 based on the ratio of an air signal to a liquid signal, where the ratio represents the ratio of the reflectivity of light relative to the body 3523 in a dry state to the reflectivity of light relative to the body 3523 in a wet state. However, embodiments of the present disclosure are not limited thereto.

[0119] For example, according to embodiments of this disclosure, controller 70 may alternatively use an "imaging" method (e.g., sensing fluorescence emitted by the sample due to excitation light) to determine the presence of air bubbles in the immersion liquid on immersion objective 3522. It is understood that higher NA objectives produce (capture) a larger signal compared to lower numerical aperture (NA) objectives. Furthermore, if air bubbles are present in the immersion objective, full NA of the objective cannot be achieved, resulting in a lower signal. According to embodiments of this disclosure, controller 70 may compare the signal from a dry immersion objective (or a dry immersion gas objective) with the signal from a wet immersion objective, and based on the signal not being increased by the expected amount, controller 70 may determine the presence of air bubbles.

[0120] For example, the sensors (e.g., first sensor 3515 or second sensor 3521) of embodiments of this disclosure can acquire air signals and liquid signals. The air signal is a first focused fluorescence image (or a feature thereof) of the sample held by the body 3523 in a dry state (hereinafter referred to as the air image signal), and the liquid signal is a second focused fluorescence image of the sample held by the body 3523 in a wet state, wherein the two images have the same features. According to embodiments, the fluorescence image can be obtained based on excitation light from a light source (e.g., light source 3510, light source 3510A, or light source 3510B) that causes the sample to emit fluorescence. The controller 70 can acquire and record the air image signal and liquid image signal from the sensors (e.g., first sensor 3515 or second sensor 3521). The controller 70 can be configured to compare the ratio of the air image signal to the liquid image signal with a predetermined value (e.g., a predetermined threshold) to detect the presence of air bubbles in the immersion liquid between the immersion objective 3522 and the body 3523 in a wet state.

[0121] For example, in a wet state, if the immersion liquid (e.g., water) is properly positioned on the immersion objective 3522 without any air bubbles, the intensity of the liquid image signal can be expected to increase compared to the liquid image signal with air bubbles present. In other words, in a wet state without air bubbles, the ratio of the air image signal to the liquid image signal can be much less than 1, while in a wet state with air bubbles, the ratio of the air signal to the liquid signal is approximately 1.

[0122] In view of the above, for a wet state, the controller 70 can determine whether air bubbles exist in the immersion liquid on the immersion objective lens 3522 based on the ratio of the air image signal to the liquid image signal. For example, the controller 70 can compare the ratio of the air image signal to the liquid image signal with a predetermined threshold to make the determination.

[0123] According to an embodiment, the controller 70 can determine (or detect) that no air bubbles are present in the immersion liquid between the immersion objective 3522 (immersion objective) and the body 3523 based on the ratio of the air image signal to the liquid image signal being less than a predetermined threshold. Then, based on the detection that no air bubbles are present on the immersion objective 3522, the controller 70 can automatically cause the automatic imaging system 3000 to image the sample (e.g., using the light source 3512 and the immersion objective 3522).

[0124] According to an embodiment, controller 70 can determine (or detect) the presence of air bubbles in the immersion liquid between immersion objective 3522 (immersion objective) and body 3523 based on the ratio of air image signal to liquid image signal being greater than or equal to a predetermined threshold. Based on the detection of air bubbles, controller 70 may not automatically perform analysis. For example, controller 70 may enable output device 20 (see...) Figure 12 The system outputs an error alert to the user so that the user can manually check for air bubbles in the immersion fluid between the immersion objective and the body 3523. If the user confirms the presence of air bubbles, the user can manually reapply the immersion fluid to the immersion objective 3522, or via input on the input device 10 (see [link]). Figure 12 The controller 70 is instructed to automatically reapply the immersion liquid to the immersion objective 3522. Afterward, the controller 70 can re-perform bubble detection. Alternatively, if the user confirms that no bubbles are present, the user can do so via input device 10 (see...). Figure 12 The input on the sensor is used to cover errors so that the controller 70 can control the analysis to be performed (e.g., capturing sample images using the light source 3512 and the immersion objective 3522).

[0125] According to embodiments of this disclosure, the predetermined threshold can be determined or obtained in various ways (e.g., by the controller 70). For example, the controller 70 can determine the predetermined threshold based on its image analysis results and / or bubble detection results, or the controller 70 can obtain the predetermined threshold by, for example, a user inputting the predetermined threshold to the controller 70 using the input device 10 (see [link to relevant documentation]). Figure 12 ).

[0126] According to embodiments of this disclosure, controller 70 can be configured to determine whether air bubbles exist in the immersion liquid between the immersion objective 3522 (immersion objective) and the body 3523 based on a liquid signal (or liquid image signal) without obtaining an air signal (or air image signal). For example, controller 70 can be configured to determine that no air bubbles exist based on a liquid signal less than a predetermined threshold, or to determine that air bubbles exist based on a liquid signal greater than or equal to a predetermined threshold. Furthermore, controller 70 can be configured to determine that no air bubbles exist based on a liquid image signal greater than a predetermined threshold, or to determine that air bubbles exist based on a liquid image signal less than or equal to a predetermined threshold. As described above, the predetermined threshold can be determined or obtained in various ways (e.g., by controller 70).

[0127] Figures 8A-8B This is a flowchart illustrating an operation method of an automatic imaging system according to an embodiment of the present disclosure.

[0128] For clarity, reference will be made to Figure 1 Description of the first embodiment shown Figures 8A-8B The methods described herein. However, those skilled in the art will understand that these methods can also be performed by other embodiments of this disclosure (including the second to ninth embodiments described above).

[0129] Reference Figure 8A The controller 70 can control the light source 3510 to emit light 3501, causing the light 3501 to travel through the dry objective lens to the body 3523 (operation 3401). According to an embodiment, the dry objective lens can be an immersion objective lens or an immersion objective lens that is not currently immersed in liquid. In this dry state, the controller 70 can measure and record the corresponding air signal level (or air image signal level) received by the first sensor 3515 due to light reflected from the body 3523 (e.g., reflected light 3502) or due to light generated by the sample based on light 3501 (operation 3402).

[0130] Then, controller 70 can control moving mechanism 3530 to move the immersion objective lens, replacing the dry objective lens, to an observation position below body 3523 (operation 3403). According to embodiments, the moved immersion objective lens can be a different objective lens from the gas or liquid immersion objective lens used in the dry state, or it can be the same immersion objective lens used in the dry state, but with an immersion liquid applied to it. Operation 3403 may also include controller 70 (or user) applying immersion liquid to the immersion objective lens before or after moving it to the observation position. In the case that the immersion objective lens is the same as the immersion objective lens used in the dry state, operation 3403 may also include controller 70 (or user) applying immersion liquid to the immersion objective lens before, after, or instead of moving it to the viewing position.

[0131] The controller 70 can control the light source 3510 to emit light 3501, such that the light 3501 travels through an immersion objective lens having an immersion liquid on it to the body 3523 (operation 3404). In this wet state, the controller 70 can measure and record the corresponding water signal level (or water image signal level) received by the first sensor 3515 due to light reflected from the body 3523 (e.g., reflected light 3502) or due to light generated by the sample based on light 3501 (operation 3405).

[0132] Then, the controller 70 can compare the ratio of the air signal (or air image signal) to the liquid signal (liquid image signal) with a predetermined threshold (operation 3406). For example, if the ratio of the air signal to the liquid signal is obtained (this ratio represents the ratio of the reflectivity of light relative to the body 3523 during dry and wet states), the controller 70 can determine that there are no air bubbles in the immersion fluid on the immersion objective lens based on the ratio being greater than the predetermined threshold (operation 3407). Alternatively, the controller 70 can determine that there are air bubbles in the immersion fluid on the immersion objective lens based on the ratio being less than or equal to the predetermined threshold (operation 3408).

[0133] In operation 3408, controller 70 can also cause output device 20 (see...) Figure 12 It outputs an alarm indicating an error to the user so that the user can manually check whether there are air bubbles in the immersion liquid between the immersion objective and the body 3523.

[0134] If the user confirms the presence of air bubbles, the user can retry forming a clump on the objective lens (operation 3410). For example, the user can manually reapply the immersion liquid to the immersion objective lens, or via input on input device 10 (see [link]). Figure 12The controller 70 is instructed to automatically reapply the immersion liquid to the immersion objective. According to an embodiment, operation 3410 may include the controller 70 (and / or the user) retracting the immersion objective, dispensing the immersion liquid onto the immersion objective, and bringing the body 3523 back into contact with the immersion objective.

[0135] Alternatively, if the user confirms that there are no bubbles, the user can input via input device 10 (see [link]). Figure 12 To cover the error, the controller 70 performs an analysis (e.g., capturing a sample image using the light source 3512 and the immersion objective) (operation 3409).

[0136] In operation 3407, when no bubbles are detected in operation 3406, controller 70 can automatically perform analysis (e.g., capturing sample images using light source 3512 and immersion objective) (operation 3407). Furthermore, in operation 3407, controller 70 can also enable output device 20 (see...) Figure 12 An alarm will be output indicating that no bubbles have been detected.

[0137] According to an embodiment, operations 3401 and 3402 can be performed by the controller 70 in a first stage (e.g., a calibration stage), and subsequent operations (e.g., operations 3404, 3403, etc.) can be performed by the control device 70 in a second stage (e.g., an operation stage). According to an embodiment, after performing a single first stage, the controller 70 can perform the second stage multiple times. In other words, the second stage can be performed multiple times based on a single first stage. For example, in the repetition of operation 3406, the controller 70 can compare the same recorded air signal (or air image signal) with multiple recorded liquid signals (or liquid image signals) to determine whether air bubbles are present in the immersion liquid on each immersion objective.

[0138] Reference Figure 8B In the case where the main body 3523 is a microplate comprising multiple samples in each well, the method may further include, after operation 3403, the controller 70 moving one of the samples to a capture position (operation 3411). For example, the controller 70 may control at least one actuator of an automated imaging system to move the microplate such that one of the samples is directly above the immersion objective lens so that it can be seen by the immersion objective lens. After performing operation 3410, operations 3404, 3405, etc., may be performed.

[0139] Furthermore, after capturing a sample image of one sample (operation 3407), the controller 70 can determine whether another sample in the microplate should be imaged (operation 3415). For example, if there is a previously unimaged sample in the microplate, the controller 70 can determine that another sample to be imaged exists. According to embodiments, the controller 70 can determine the presence of another sample to be imaged based on a count of the number of imagesd samples relative to the total number of samples, or based on the sensor output sensing the position of the orifices of the microplate relative to the immersion objective, but embodiments of this disclosure are not limited thereto.

[0140] When imaging another sample, controller 70 can move the next sample to the capture position (operation 3413). For example, controller 70 can control at least one actuator of the automated imaging system to move the microplate so that the next sample is directly above the immersion objective and can be seen by the immersion objective. Operations 3405 and 3406 can then be repeated.

[0141] When there are no more samples to image, controller 70 can determine that the imaging process is complete and eject the sample (operation 3414). For example, controller 70 can control at least one actuator of the automated imaging system (or enable the user) to move the microplate away from the immersion objective.

[0142] Reference Figure 12 The automatic imaging system 1 disclosed herein (e.g., automatic imaging systems 3000 and 3000A-H) may include an input device 10, an output device 20, a light source 30, a sensor 40, an actuator 50, and a controller 70.

[0143] Input device 10 may include, for example, a microphone, keyboard, mouse, switch, button, and / or a touchscreen display. Output device 20 may include, for example, a speaker, display, and / or a piezoelectric buzzer. Light source 30 may include, for example, the light sources described above in the embodiments of this disclosure (e.g., light source 3510, light source 3510A, light source 3510B, light source 3512, light source 3152A, light source 3518, etc.). Sensor 40 may include, for example, the sensors described above in the embodiments of this disclosure (e.g., first sensor 3515, second sensor 3521, etc.). Actuator 50 may include, for example, the actuators described above that move various components of the embodiments of this disclosure.

[0144] The controller 70 may include hardware and / or software components capable of automatically controlling any number (e.g., all or some) of the components of the automatic imaging system 3000 to perform their respective functions, wherein the automatic control may be based on one or more user inputs to the automatic imaging system 3000. For example, the controller 70 may include at least one processor 4501 and a memory 74. The controller 70 may be connected to an input device 10 for receiving input from it, an output device 20 for sending output to it, a light source 30 for controlling the emission of a light source 30, a sensor 40 for receiving information therefrom (e.g., air and water signals), and an actuator 50 for controlling the components. According to an embodiment, the memory 74 may store computer instructions. When executed by at least one processor 4501, the computer instructions may be configured to cause the controller 70 to perform its functions. According to an embodiment, the controller 70 may be wired and / or wirelessly connected to one or more (e.g., some or all) of the automatic imaging system 3000 configured as controlled components (e.g., output device 20, light source 30, sensor 40) for controlling these components. According to an embodiment, the controller 70 can be connected via wired and / or wireless means to one or more (e.g., some or all) of the light / image sensing components (e.g., sensor 40) of the automatic imaging system 3000 to receive light / image related signals.

[0145] According to embodiments of this disclosure, the controller 70 may include a hardware communication interface configured to connect the automated imaging system 3000 to an external device (e.g., a computer, mobile device, etc.) via wired and / or wireless means. For example, the hardware communication interface may be configured to connect at least one processor 72 and memory 74 of the automated imaging system to the external device via a local area network and / or a wide area network (e.g., the Internet). Through the hardware communication interface, at least one processor 72 and memory 74 may be configured to report information to the external device regarding the status, results, and / or schedule of sample analysis of the automated imaging system. According to embodiments, through the hardware communication interface, at least one processor 72 and memory 74 may be configured to receive commands from external components to control components of the automated imaging system, including the scheduling of sample analysis. At least one processor 72 and memory 74 may be configured to perform control based on commands.

[0146] The methods and systems described above can be implemented using hardware, software, and any combination of hardware and software. For example, the systems described above can be implemented as including one or more processors (e.g., CPU, microcontroller, microprocessor, etc.) that execute computer-readable instructions stored in computer-readable memory (e.g., ROM, RAM, flash memory, etc.). The computer-readable instructions can be provided as machine-readable algorithms that result in the execution of the algorithms discussed above regarding the systems and flowcharts.

[0147] Embodiments of this disclosure allow an automated imaging system to easily detect the presence of air bubbles in the immersion liquid between the objective lens and the body configured to hold the sample before the automated imaging system captures an image of the sample.

[0148] The embodiments disclosed herein can achieve the advantages described herein. It should also be understood that various modifications, adaptations, and alternative embodiments thereof are included within the spirit and scope of this disclosure.

Claims

1. An automatic imaging system, comprising: Immersion objective lens; A light source configured to emit first light toward the body via the immersion objective while the body is positioned on the immersion objective between the immersion objective and the body, wherein the body is configured to hold the sample; A first sensor, configured to receive a first signal based on the first light after the first light reaches the body via the immersion objective; and A controller configured to detect the presence of air bubbles in the immersion liquid between the immersion objective and the body based on the first signal received by the first sensor.

2. The automatic imaging system according to claim 1, further comprising: Immersion objective lens, The light source is further configured to emit a second light toward the body via the immersion objective lens. The first sensor is further configured to receive a second signal based on the second light after the second light reaches the body via the immersion objective, and The controller is also configured to detect the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the first signal and the second signal received by the first sensor.

3. The automatic imaging system according to claim 2, wherein, The controller is also configured to detect the presence of the air bubbles in the immersion liquid between the immersion objective and the body by comparing the ratio of the second signal to the first signal with a predetermined threshold.

4. The automatic imaging system according to claim 3, wherein, The controller is also configured to: Based on the fact that the ratio of the second signal to the first signal is greater than the predetermined threshold, it is detected that there are no air bubbles in the immersion liquid between the immersion objective and the main body; and The sample is analyzed based on the detection that no air bubbles are present in the immersion liquid between the immersion objective and the body.

5. The automatic imaging system according to claim 3, wherein, The controller is also configured to detect the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being less than or equal to the predetermined threshold.

6. The automatic imaging system according to claim 2, wherein, At least a portion of the first light emitted from the light source and at least a portion of the second light are reflected by the subject, and The first sensor is configured to receive the first signal based on the portion of the first light reflected by the subject, and to receive the second signal based on the portion of the second light reflected by the subject.

7. The automatic imaging system according to claim 2, wherein, The first light is a first excitation light configured to cause the sample to emit a third fluorescence light, and the second light is a second excitation light configured to cause the sample to emit a fourth fluorescence light. The first sensor is configured to receive the first signal based on the third light emitted by the sample, and to receive the second signal based on the fourth light emitted by the sample.

8. The automatic imaging system according to claim 1, wherein, The light source is a narrow-beam light source that includes a laser diode.

9. The automatic imaging system according to claim 1, wherein, The first sensor is an image sensor.

10. The automatic imaging system according to claim 1, wherein, The first sensor is adjacent to the light source, and The automated imaging system also includes a second sensor, which is an image sensor configured to image the sample.

11. The automatic imaging system according to claim 1, wherein, The light source is a wide-beam light source that includes lamps.

12. The automatic imaging system according to claim 1, further comprising: Rotary confocal microscope The rotating confocal microscope includes: The liquid-immersed objective lens; The light source; The first sensor; Confocal turntable; At least one first optical body defining a first infinity correction optical region on a first side of the body oriented to hold the sample on the confocal turntable; and At least one second optical body defines a second infinity correction optical region on a second side of the body, which is remotely configured to hold the sample, on the confocal turntable.

13. The automatic imaging system according to claim 12, wherein, The light source and the first sensor are located in the first infinity-corrected optical region of the turntable confocal microscope.

14. The automatic imaging system according to claim 12, wherein, The light source and the first sensor are located in the second infinity correction optical region of the turntable confocal microscope.

15. The automatic imaging system according to claim 1, wherein, The light source is further configured to emit a second light toward the subject via the immersion objective when there is no immersion liquid on the objective lens, and The first sensor is further configured to receive a second signal based on the second light after the second light reaches the body via the immersion objective lens, and The controller is also configured to detect the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the first signal and the second signal received by the first sensor.

16. A method performed by an automated imaging system, the method comprising: With the liquid submerged between the immersion objective of the automated imaging system and the main body of the automated imaging system, and the light source of the automated imaging system emitting a first light through the immersion objective onto the main body, wherein the main body is configured to hold the sample; After the first light reaches the subject via the immersion objective, the first sensor of the automated imaging system receives a first signal based on the first light; and Based on the first signal received by the first sensor, the presence of air bubbles in the immersion liquid between the immersion objective and the body is detected.

17. The method of claim 16, further comprising: The light source emits a second light toward the subject via the gas immersion objective of the automatic imaging system; and After the second light reaches the body via the immersion objective, the first sensor receives a second signal based on the second light. The detection includes detecting the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the first signal and the second signal received by the first sensor.

18. The method according to claim 17, wherein, The detection includes detecting the presence of the air bubbles in the immersion liquid between the immersion objective and the body by comparing the ratio of the second signal to the first signal with a predetermined threshold.

19. The method according to claim 18, wherein, The detection includes detecting that no air bubbles exist in the immersion liquid between the immersion objective and the body, based on the ratio of the second signal to the first signal being greater than the predetermined threshold. The method further includes analyzing the sample based on the detection that no air bubbles are present in the immersion liquid between the immersion objective and the body.

20. The method according to claim 18, wherein, The detection includes detecting the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the ratio of the second signal to the first signal being less than or equal to the predetermined threshold.

21. The method of claim 17, further comprising: At least a portion of the first light and at least a portion of the second light emitted from the light source are reflected by the subject. Receiving the first signal includes receiving the first signal based on the portion of the first light reflected by the subject, and Receiving the second signal includes receiving the second signal based on the portion of the second light reflected by the subject.

22. The method according to claim 17, wherein, The emission of the first light causes the sample to emit a third light via fluorescence. The second light is emitted, causing the sample to emit a fourth light via fluorescence. Receiving the first signal includes receiving the first signal based on the third light emitted by the sample, and Receiving the second signal includes receiving the second signal based on the fourth light emitted by the sample.

23. The method of claim 16, further comprising: When there is no immersion liquid on the immersion objective lens, a second light is emitted from the light source towards the main body via the immersion objective lens; After the second light reaches the body via the immersion objective, the first sensor receives a second signal based on the second light. The detection includes detecting the presence of the air bubbles in the immersion liquid between the immersion objective and the body based on the first signal and the second signal received by the first sensor.

24. A non-transitory computer-readable medium storing computer instructions, which, when executed by at least one processor, cause the at least one processor to: With the liquid immersed between the immersion objective lens and the main body of the automated imaging system, and the light source of the automated imaging system emitting first light towards the main body via the immersion objective lens, wherein... The main body is configured to hold the sample; After the first light reaches the subject via the immersion objective lens, the first sensor of the automatic imaging system receives a first signal based on the first light. and Based on the first signal received by the first sensor, the presence of air bubbles in the immersion liquid between the immersion objective and the body is detected.

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