Fluid immersion control for inverted microscopy

Abstract

A fluid immersion control system can monitor the electrical resistance of the fluid as an indication of the presence of a fluid layer having a meniscus greater than the diameter of the optical axis for immersion microscopy using a common electrode and a plurality of sensor electrodes at a planar surface associated with a distal end of an immersion microscope objective. The fluid immersion control system can activate replenishment of the fluid when the electrical resistance indicates that the diameter is not immersed in the fluid.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 969,809, filed February 4, 2020, pursuant to 35 U.SC §119(e), which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This disclosure relates generally to inverted microscopy, and more specifically, to fluid immersion control for inverted microscopy. Background Technology

[0004] Inverted microscopy refers to the arrangement of microscopic components and the optical path that is inverted or reversed from that of a typical tabletop microscope. Specifically, inverted microscopy involves the use of a sample stage or table with a transparent underside, beneath which the microscope objectives are pointed upwards to image the sample through the transparent underside. In this way, multiple samples, such as samples in a one-dimensional or two-dimensional array, can be placed on the sample stage and simultaneously introduced into the inverted microscope for imaging. The inverted microscope (or sample stage) can be equipped with a motion control system, such as a dual-axis (or stage) motion controller, which, for example, allows each individual sample among multiple samples to be positioned above the microscope objectives for imaging.

[0005] Inverted microscopy, due to its ability to simultaneously prepare and introduce large numbers of samples, has been used for in vitro diagnostics and other high-throughput examinations of biological samples, and is suitable for both qualitative and quantitative analysis. Specifically, inverted microscopy has been used for analyses involving gene sequencing, as well as for high-throughput imaging of biological samples such as tissues, cell cultures, and biological fluids. The need for bioanalyses involving higher spatial resolution or longer depth of field imaging of live samples has led to the use of optical systems that operate using a fluid medium in physical contact between the inverted microscope objective and the sample focal plane (e.g., on the transparent underside of the sample stage). The choice of a fluid medium with a higher refractive index than air increases the numerical aperture (NA) of the inverted microscope objective, thereby increasing the resolution of the inverted microscope compared to imaging in air. Therefore, this high-NA optical system relies on the integrity of the physical contact of the fluid medium between the inverted microscope objective and the sample focal plane to achieve high-throughput imaging.

[0006] An example of inverted microscopy is provided by U.S. Patent No. 8,199,407B2, issued June 12, 2012, entitled “Immersion Object, Apparatus for Forming an Immersion Film and Method,” which is incorporated herein by reference as if it were set forth herein in its entirety. Attached Figure Description

[0007] To gain a more complete understanding of the invention and its features and advantages, reference is now made to the following description in conjunction with the accompanying drawings, wherein:

[0008] Figure 1 Selected elements of the inverted microscope system are depicted;

[0009] Figure 2 Selected components of the fluid immersion control system are described;

[0010] Figure 3 The selected components of the measurement circuit are described;

[0011] Figure 4 depicts a prior art inverted microscope objective in motion;

[0012] Figure 5 An inverted microscope objective with immersion fluid control in motion is depicted;

[0013] Figure 6 Details of the sensor ring mounted to the microscope objective are depicted;

[0014] Figure 7 A detailed top view of the sensor ring is provided;

[0015] Figure 8 A detailed bottom view of the sensor ring is provided;

[0016] Figure 9 Further details are depicted in the cross-sectional view of the sensor ring;

[0017] Figure 10 Further details are depicted in the cross-sectional view of the sensor ring mounted to the microscope objective;

[0018] Figure 11 The working distance is depicted in a cross-sectional view of the sensor ring mounted on the microscope objective.

[0019] Figure 12 This is a flowchart of the inverted immersion microscopy procedure;

[0020] Figure 13 Another embodiment in which the sensor ring is formed within an integrated microscope objective body is depicted;

[0021] Figure 14 Another embodiment of the sensor ring is described; and

[0022] Figure 15 Another embodiment in which the sensor ring is attached to the body of the microscope objective is depicted. Detailed Implementation

[0023] In the following description, details are illustrated by way of example to facilitate discussion of the disclosed subject matter. However, it should be clear to those skilled in the art that the disclosed embodiments are exemplary and not exhaustive of all possible embodiments.

[0024] Throughout this disclosure, the hyphenated form of the reference numerals refers to a specific instance of an element, while the non-hyphenated form refers to an element in general or collectively. Thus, by way of example (not shown in the figures), device "12-1" refers to an instance of a class of devices that can be collectively referred to as device "12," and any one of them can generally be referred to as device "12." In the figures and descriptions, the same reference numerals are intended to denote the same elements.

[0025] As previously mentioned, the need for bioanalysis involving higher spatial resolution or longer imaging depth of living samples has led to the use of optical systems that operate using a fluid medium in physical contact between the inverted microscope objective and the sample focal plane. For example, the sample focal plane may be located on a transparent underside of the sample stage, such as a slide used for in vitro diagnostics.

[0026] In various embodiments, multiple individual samples can be placed in a defined array with a transparent underlayer, such as a well plate or a similar support for multiple samples, and can be scanned individually using an inverted microscope objective and a motion control system. The motion control system can be made capable of moving the samples relative to the inverted microscope objective to observe and image each individual sample. In some embodiments, the sample stage can be moved relative to the inverted microscope objective. In yet another embodiment, at least one of the sample stage and the inverted microscope objective can be moved relative to each other. In any case, the motion control system can perform individual movements to access individual samples. For example, a movement starting from the first sample can involve acceleration, translation at a given speed, and deceleration to stop at the second sample. Furthermore, individual movements can involve changes in at least one direction. Note that other types of continuous motion profiles, such as those without a constant velocity portion, can be used in various embodiments. Therefore, when using immersion microscopy, the fluid used for immersion may be lost or depleted during acceleration, translation, or deceleration. Specifically, the fluid can recede from the diameter of the optical axis defined by the inverted microscope objective, such as the cross-sectional area of ​​the optical portion of the inverted microscope objective (see also prior art Figure 4). When the fluid does not fully immerse the diameter of the optical axis, then the numerical aperture of the inverted microscope objective for the non-immersed portion is not increased, and the benefits of immersion microscopy cannot be realized.

[0027] Therefore, when fluid is lost during transport, it should be replenished before immersion microscopy can be performed. In conventional systems, once the microscope stops and the sample is being imaged, the loss of fluid can be detected and the corresponding fluid refilled. Therefore, replenishing fluid at the imaging location can consume significantly additional sampling time for each individual sample, which is undesirable and can adversely affect the overall economic efficiency of the entire microscopy system (e.g., by limiting maximum sample throughput). In particular, for sample arrays with a large number of samples, the increased analysis time due to replenishing fluid at the imaging location can be substantial and significant. It should be noted that fluid can also be lost during operation due to other factors such as evaporation, humidity levels, temperature or pressure changes, and others.

[0028] As disclosed herein, systems and methods for fluid immersion control in inverted microscopy are disclosed, capable of maintaining fluid along a diameter corresponding to the optical axis of an inverted microscope objective. The systems and methods for fluid immersion control in inverted microscopy provide a sensor ring that contacts the microscope objective body and forms an annulus through which fluid can be replenished. Even when the fluid no longer completely immerses the diameter of the optical axis of the inverted microscope objective, a common electrode of the sensor ring can be located within the annulus where the common electrode remains in contact with the fluid. In addition to the common electrode, the sensor ring includes a plurality of sensor electrodes circumferentially arranged around the diameter of the optical axis. In a particular embodiment, eight sensor electrodes can be arranged in a circle slightly larger than the diameter of the optical axis, and can be able to detect any depletion of fluid along that diameter.

[0029] Furthermore, the system and method disclosed herein for fluid immersion control in inverted microscopy can provide a sensor ring that forms a substantially flat surface when attached to an inverted microscope objective, such that the sensor electrodes and the distal end of the inverted microscope objective are located on a flat surface to which one surface of the meniscus of the immersion fluid is attached. As a result of this coplanar arrangement, it is noteworthy that signals from the sensor electrodes can reliably detect fluid or any missing portion of the fluid without any geometric complexity that could affect the location of the fluid contact with the microscope.

[0030] The system and method for fluid immersion control in inverted microscopy disclosed herein can provide a fluid immersion control system that can adjust the amount of fluid used for immersion. Specifically, as described above, the fluid immersion control system can, for example, continuously measure the resistance of the fluid from each sensor electrode simultaneously during movement to different samples. In this way, the system and method for fluid immersion control in inverted microscopy disclosed herein can provide high sensitivity in situations where the fluid is depleted or partially depleted from the diameter of the optical axis. High sensitivity can be sensitivity to changes in fluid level as well as sensitivity to depletion time and replenishment time. Therefore, the system and method for fluid immersion control in inverted microscopy disclosed herein can, for example, rapidly detect even at least partially depleted fluid levels during scanning motion, and can rapidly respond and timely replenish fluid for the next sample, such that immersion microscopy imaging can be performed immediately without delay when the inverted microscope objective stops at the next sample.

[0031] Now refer to the attached diagram, Figure 1 An inverted microscope system 100 for performing immersion microscopy is depicted. Note that the inverted microscope system 100 is... Figure 1 The illustrations are schematic and may not be drawn to scale or in perspective. In particular, note that... Figure 1The various elements and components shown can be used in different implementations of the inverted microscope system 100.

[0032] like Figure 1 As shown, the inverted microscope system 100 uses a fluid layer 106 between the inverted microscope objective 102 and the sample plate 108. The fluid layer 106 can form a meniscus 106-1 between the flat surfaces of the sensor ring 104 mounted to the distal end of the inverted microscope objective 102. Note that various types of fluids or fluid mixtures can be used for the fluid layer 106, and the working distance between the sensor ring 104 and the sample plate 108 can be controlled by certain physical properties of the fluid layer 106, such as the surface tension of the fluid layer 106 (see also...). Figure 11 In a particular embodiment, the fluid layer 106 has a higher refractive index than air. In one embodiment, the fluid layer 106 comprises deionized water. Although the sample plate 108 is shown as a monolithic structure, it is noted that the sample plate 108 itself may be a single element among multiple elements, such as a single slide among multiple slides for imaging, either individually or collectively.

[0033] exist Figure 1 In the figure, coordinate axis 116 defines the direction of the XY plane that is parallel to the sample plate 108 and perpendicular to the Z-axis, which is parallel to the optical axis 114. As shown in the figure, optical axis 114 represents the optical axis of the inverted microscope objective 102, and optical axis 114 extends through the first diameter at the distal portion 102-1 of the inverted microscope objective 102 (in Figure 1 Not visible in the middle, see also Figure 6 , Figure 9 (The diameter is 906). Specifically, light enters the inverted microscope objective 102 in direction 117 along the optical axis 114 (corresponding to the Z-axis of coordinate axis 116). As shown, the light source 112 is used to illuminate multiple samples 110 located on the sample plate 108 in direction 117. Samples 110 are schematic and can correspond to any kind of liquid, solid, or mixture that can be imaged using the inverted microscope objective 102, for example, including biological samples with different cellular structures and chemical substances. Figure 1In this configuration, the second sample 110-2 is illuminated by the light source 112, while the first sample 110-1 and the third sample 110-3 are adjacent to the second sample 110-2, representing the sample array carried by the sample plate 108. While the light source 112 is typically designed to output visible light frequencies, it is important to note that the light source can generate light of various frequencies and can generate coherent or incoherent light. Therefore, the sample plate 108 can be constructed of a material transparent to at least some of the light frequencies generated by the light source 112, such as glass or a transparent polymer. It is also important to note that the sample plate 108 and the samples 110 can represent various structures and supports for multiple samples, such as well plates or other types of supports that can hold and image the samples using the inverted microscope system 100.

[0034] exist Figure 1 In the schematic diagram, the optical axis 114 continues through the inverted microscope objective 102 and into the optical microscope 120, which can represent various types and arrangements of optical microscopes or components capable of imaging, including quantitative and qualitative analysis. Figure 1 Also shown is a stage control system 122 that can represent various components of a motion control system, which is capable of moving at least one of the sample plate 108 and the inverted microscope objective 102 relative to each other, as previously described, for example, to analyze multiple samples 110.

[0035] Figure 1 The fluid immersion control system 150 is also shown, which will be described in more detail below, and which may include or may be able to support the operation of the sensor ring 104. Figure 1 The diagram also labels fluid interface 154 and electronic interface 152 connecting the fluid immersion control system 150 to the sensor ring 104. Fluid interface 154 may represent a conduit in fluid communication with the sensor ring 104 to supplement the fluid layer 106, while electronic interface 152 may represent an electrical connection to a common electrode and sensor electrodes included in the sensor ring 104. Further details of the fluid immersion control system 150 are described below regarding... Figure 2 Describe it.

[0036] In operation of the inverted microscope system 100, a sample plate 108 and multiple samples 110 can be introduced for imaging analysis. A stage control system 122 is operable to translate the inverted microscope objective 102 relative to the sample plate 108 in the XY plane to image individual samples 110, such as those shown for sample 110-2. Furthermore, a fluid immersion control system 150 can be activated and can automatically introduce and maintain a fluid layer 106 between the distal portion 102-1 of the inverted microscope objective 102 and the sample plate 108, including during movement. Therefore, the fluid immersion control system 150 ensures that immersion microscopy can be performed using the inverted microscope objective 102 at any desired time without significant time limitations or limitations associated with the movement controlled by the stage control system 122. In this way, the inverted microscope system 100, as shown and described above, can improve the efficiency and productivity of inverted immersion imaging.

[0037] Now for reference Figure 2 Further details of the fluid immersion control system 150 are depicted in the schematic diagram. Therefore, Figure 2 It may not be drawn to scale or in perspective. Figure 2 In the diagram, the fluid immersion control system 150 is shown to include a sensor interface 204, an immersion controller 206, and a fluid replenishment system 208. Specifically, the sensor interface 204 is coupled to the sensor ring 104 via an electronic interface 152, which may include multiple signal lines or other connectors to enable the measurement circuit 300 (see [link to diagram]). Figure 3 It can be closed. Furthermore, the fluid replenishment system 208 is coupled to the sensor ring 104 via a fluid interface 154, which may be a conduit coupled to and in fluid communication with internal passages within the sensor ring 104, as described below for example... Figure 9 and Figure 10 Further detailed description.

[0038] like Figure 2 As shown, the immersion controller 206 can provide processing and logic functions to interpret measurement signals and generate drive signals accordingly. Therefore, the immersion controller 206 may internally include a processor and a memory medium capable of storing and providing executable code to the processor, as well as other circuitry and components. In some embodiments, the immersion controller 206 may include a field-programmable gate array (FPGA) that implements at least some of the logic functions of the fluid immersion control system 150. In a particular embodiment, the immersion controller 206 may be an embedded controller capable of operating autonomously without user input, such as operating in a dedicated automated manner. It should also be understood that the fluid immersion control system 150 may rely on at least one power source (not shown), which may be... Figure 2The external or internal power source of the component depicted.

[0039] In such Figure 2 In the fluid immersion control system 150 shown, the sensor interface 204 can realize and use the measurement circuit 300 (see...). Figure 3 To perform resistance measurements of the fluid layer 106 used for immersion microscopy, as per... Figure 1 As described in detail. Therefore, sensor interface 204 can provide power, such as a voltage source or current source 302 (see [reference]). Figure 3 This power supply provides power to the measurement circuit 300. Note that in various embodiments, the power supply may operate in DC or AC mode. Furthermore, the sensor interface 204 can provide signal conditioning, amplification, and digitization to the input signal from the measurement circuit 300. In different embodiments, various means and methods for signal conditioning, amplification, and digitization can be used. For example, when a single analog-to-digital converter (ADC, not shown) is used with the sensor interface 204, the sensor interface 204 may include a multiplexer (not shown) to sequentially switch and digitize signals such as those from sensor electrode 306 and common electrode 304 (see [link to documentation]). Figure 3 The sensor interface 204 may include a plurality of corresponding input signals. In other embodiments, the sensor interface 204 may include a plurality of ADCs corresponding to each of the plurality of input signals. In various embodiments, the sensor interface 204 may have a digital interface corresponding to the immersion controller 206 and may send measurements or other indications of the results of performed measurements to the immersion controller 206. The results of the measurements may indicate whether the amount of fluid in the fluid layer 106 is sufficient for immersion microscopy.

[0040] In such Figure 2 In the fluid immersion control system 150 shown, the immersion controller 206 can correspondingly receive measurement results from the sensor interface 204 and can decide whether to activate the fluid replenishment system 208 to refill the sensor ring 104 with fluid. In some embodiments, the immersion controller 206 can directly control the fluid replenishment at any time and can directly control the flow of fluid through the fluid interface 154. In some embodiments, the fluid replenishment system 208 can have some degree of control, such as enabling fluid to flow through the fluid interface 154 for a period of time in response to a single instruction from the immersion controller 206.

[0041] In such Figure 2In the fluid immersion control system 150 shown, the fluid replenishment system 208 may include various components to provide fluid via the fluid interface 154 in response to an instruction from the immersion controller 206. Therefore, the fluid replenishment system 208 may include a tank or reservoir (not shown) for a given volume of fluid. The internal reservoir of the fluid replenishment system 208 may be in fluid communication with a pump (not shown), which in turn is in fluid communication with the fluid interface 154 to output fluid.

[0042] exist Figure 3 The measurement circuit 300, as described above, is depicted in the figure. As shown, the measurement circuit is used for resistance measurement of the fluid layer 106 between the individual sensor electrodes 306 and the common electrode 304, and also includes a current source 302 and a switch 308. Each sensor electrode 306 can be formed as an outer surface electrode at the working surface of the sensor ring 104, as will be shown and described in further detail below. The sensor electrodes can be shaped into an arc and can surround the diameter 906 of the optical axis 114 of the inverted microscope objective 102 (see Figure 100). Figure 9 The resistors are arranged circumferentially. As shown in the measurement circuit 300, resistors R-1, R-2, R-3, R-4, R-5, R-6, R-7, and R-8 correspond to the resistances of the fluid layer 106, respectively measured by eight sensor electrodes 306. Resistor R-1 is measured by sensor electrode 306-1, resistor R-2 by sensor electrode 306-2, resistor R-3 by sensor electrode 306-3, resistor R-4 by sensor electrode 306-4, resistor R-5 by sensor electrode 306-5, resistor R-6 by sensor electrode 306-6, resistor R-7 by sensor electrode 306-7, and resistor R-8 by sensor electrode 306-8. Switch 308 is shown as being able to be individually connected to sensor electrode 306 and, in an exemplary state, is shown in the position where sensor electrode 306-2 in the measurement circuit 300 is switched to measure resistor R-2. Note that other means for separately measuring the resistance of sensor electrode 306 can be used in different embodiments. Although current source 302 is shown as corresponding to a voltage measurement (not shown) at sensor electrode 306-2 for measuring resistance, it should be understood that other arrangements for resistance measurement, such as voltage sources and current measurements, can be used.

[0043] In the operation of the measuring circuit 300, for example, each electrode 306 can be switched individually for measurement using switch 308. The measuring circuit 300 can continue monitoring when the resistance values ​​from all electrodes 306 indicate the presence of fluid. When the resistance value from at least one electrode 306 indicates the absence of fluid, the measuring circuit 300 can instruct the addition of fluid.

[0044] Referring now to Figure 4, a prior art depiction is shown of the inverted microscope objective 408 during movement from above through a transparent sample plate (not visible in Figure 4). Visible in Figure 4 is a spacer ring 404 showing the diameter 906 of the optical axis of the inverted microscope objective 408. As the inverted microscope objective 408 moves, such as from a first sample to a second sample as described above, the meniscus 402 of the fluid layer used for immersion microscopy is shown to dynamically recede and no longer cover the spacer ring 404, which is not applicable to inverted microscopy imaging using the inverted microscope objective 408.

[0045] exist Figure 5 The diagram shows a similar depiction to that in Figure 4, but with the elements of an inverted microscope system 100 for fluid immersion control as described herein, including an inverted microscope objective 102, a sensor electrode 306, and a sensor ring 104. Figure 5 The diameter 906 of the optical axis of the inverted microscope objective 102 is shown in the image (see [reference]). Figure 9 ) ring 504. In Figure 5 In the process, when the inverted microscope objective 102 moves, due to the operation of the fluid immersion control system 150, the meniscus 106-1 maintains a diameter larger than that of the ring 504 on the ring 504, which allows immersion microscopy to be performed without interruption or time or motion constraints.

[0046] Now proceed to Figure 6 The image shows the sensor ring 104 and the inverted microscope objective 102 assembly 600, assembled in perspective view above and exploded view below. Figure 6 In the middle, the section line 900 defines the following about Figure 9 and Figure 10 The presented cross-sectional view. In, for example, an assembled... Figure 6 In the perspective view, the surface 602 of the sensor ring 104 is visible. When the sensor ring 104 is assembled with the inverted microscope objective 102, the surface 602 of the sensor ring 104 forms the working surface of the inverted microscope objective 102 for immersion microscopy, as shown. The common surface 602 is shown as substantially flat and thus facilitates the stable formation of the meniscus 106-1. Also visible in the assembly 600 is a groove 606, which is formed to carry signal traces for the respective signal electrodes 306 at the surface 602. At least one screw 608 can pass through at least one mounting hole 604 to attach the sensor ring 104 to the inverted microscope objective 102. Figure 6As shown, five screws 608 are used in conjunction with five mounting holes 604. As shown in the exploded view, a sealing ring 608 is visible, which seals the annular band 610 formed during the assembly of the sensor ring 104 with the inverted microscope objective 102, and the fluid port 612 at the radial edge of the sensor ring 104. As described herein, the annular band 610 is in fluid communication with the fluid port 612 to supply fluid to the fluid layer 106 (…). Figure 6 (Not shown in the image) provides fluid.

[0047] Figure 7 and Figure 8 Top view 104-1 and bottom view 104-2 of sensor ring 104 are depicted. In top view 104-1, various elements of sensor ring 104 are visible, including sensor electrode 306, through hole 604, and groove 606. In bottom view 104-2, the surface of sensor ring 104 forming ring band 610 is visible and includes common electrode 304, which is formed as a loop within ring band 610 to maintain contact with fluid at all times, and includes connector lead 304-1 for connection via electronic interface 152. Also visible in bottom view 104-2 is fluid port 612, which terminates within ring band 610 to replenish fluid for connection with fluid interface 154.

[0048] Figure 9 and Figure 10 Two versions, 900-1 and 900-2, are depicted respectively as cross-sectional views 900 of the sensor ring 104. Figure 9 In the cross-sectional view 900-1, the sensor ring 104 is depicted separately, while... Figure 10 In the middle section, cross-sectional view 900-2 depicts the sensor ring 104 assembled with the distal portion 102-1 of the inverted microscope objective 102 (corresponding to component 600, see [reference]). Figure 6 In cross-sectional views 900-1 and 900-2, various elements of the sensor ring 104 are visible, including the sensor electrode 306, through-hole 604, groove 606, common electrode 304, and fluid port 612. Figure 10 In the cross-sectional view 900-2, the magnified portion 902 is shown below, providing further details of the fluid port 612 and the annular band 610.

[0049] exist Figure 11 The image shows a cross-sectional view 900-3 of the sensor ring 104 assembled to the distal portion 102-1 of the inverted microscope objective lens 102. Cross-sectional view 900-3 is compared with... Figure 10The cross-sectional view 900-2 shown is substantially similar. However, in cross-sectional view 900-3, line 904 represents the working distance of the inverted microscope objective 102, such as the focal length of the inverted microscope objective 102. Therefore, the distance between surface 602 and line 904 relative to the size of sensor ring 104 can represent the working thickness of the fluid layer 106 maintained during immersion microscopy, as disclosed herein. Furthermore, the diameter 906 of the optical axis of the inverted microscope objective 102 is also... Figure 11 It is drawn in the middle.

[0050] Now for reference Figure 12 This document describes a flowchart of selected elements of an embodiment of method 1200 for fluid immersion control using inverted immersion microscopy as described herein. Method 1200, as described above, can be performed using a fluid immersion control system 150 with a sensor ring 104. Note that some operations described in method 1200 may be optional or may be rearranged in different embodiments.

[0051] Method 1200 may begin at step 1202 by observing a first sample included among a plurality of samples loaded into an inverted microscope using a microscope objective, wherein the microscope objective is immersed in fluid at its distal end. At step 1204, as the microscope objective and at least one of the first samples are moved relative to each other, an immersion controller coupled to a plurality of sensor electrodes, a common electrode, and a fluid replenishment system is used to maintain the fluid along the diameter of the optical axis of the distal end. At step 1206, the microscope objective and at least one of the first samples are moved relative to each other to observe a second sample included among the plurality of samples using the microscope objective. At step 1208, the fluid is maintained along the diameter of the optical axis before the microscope objective is aligned with the second sample.

[0052] Figure 15 Another embodiment in which the sensor ring is attached to the body of the microscope objective is depicted.

[0053] Now for reference Figure 13 Another embodiment in which the sensor ring is formed within an integrated microscope objective body is depicted. Figure 13 The image depicts the distal portion of an integrated microscope objective body. The integrated microscope objective body is formed as a single, integral workpiece and may include internal channels or conduits, as depicted. These internal channels or conduits may be routed along different paths within the integrated microscope objective body. In some embodiments, the integrated microscope objective body can be formed using any one or more suitable materials, including metals, polymers, and composite materials, employing additive manufacturing techniques such as 3D printing. Figure 13As shown, the integrated microscope objective body may also include optical components (such as microscope objective lenses and other elements associated with the optical path) and sensors for immersion control (including sensors as described above). Therefore, as shown in the figure, Figure 13 The integrated microscope objective body may include a fluid supply port that is in fluid communication with the interior of a meniscus fluid port, which may extend circumferentially around the surface of the microscope objective lens, either partially or completely. Furthermore, Figure 13 The integrated microscope objective body may include internal conduits or conductors for connection to and / or to provide a sensor interface for immersion control. The sensor interface can therefore support independent connection of one or more electrical contacts to the integrated microscope objective body and allows the sensor element to be externally connected to appropriate signal conditioning and power supplies, as explained in detail above.

[0054] An integrated microscope objective body may include a control circuitry coupled to a sensor and an analysis system capable of monitoring the sensor and detecting appropriate addition of fluid and / or cessation of imaging, and providing and / or receiving appropriate control signals and information. Alternatively, the integrated microscope objective body may include electrical contacts and / or data connections for easy connection to an analysis system and / or other components, such as a computer system or display system. For example, the integrated microscope objective body may have a USB port and / or a USB male connector for connection to the sensor and / or control circuitry within the integrated objective body, thereby allowing easy connection to both the analysis system for assembly, repair, or replacement, and also to a computer or display system.

[0055] Now for reference Figure 14 This describes another embodiment in which an integrated sensor ring is attached to the body of a microscope objective. Figure 14 The image depicts the distal portion of a microscope objective body with an integrated sensor ring attached. As illustrated, the integrated sensor ring may include all elements for immersion control, including internal channels or conduits for fluid. These internal channels or conduits may be routed along different paths within the integrated sensor ring. Figure 14As shown, the fluid conduit and sensor can be completely enclosed or embedded within the integrated sensor ring, resulting in no fluid contact between the objective body and the cover due to the annular space of the integrated sensor ring between the fluid port and the objective body. Preventing fluid contact with the objective body offers advantages compared to systems that include fluid contact between the two, such as better fluid control, easier sealing of the integrated sensor ring to the objective body, and cleaning or maintenance benefits. In some embodiments, the conduit or internal channel can be positioned directly adjacent to the objective body, such that the annular space for fluid is formed between the inner wall of the integrated sensor ring and the outer wall of the objective body. In some embodiments, the integrated sensor ring can be formed using any one or more of a variety of suitable materials, including metals and polymers, as well as composite materials, using additive manufacturing techniques such as 3D printing. Figure 14 As shown, the integrated sensor ring can be attached to the microscope objective body in a manner similar to the sensor ring previously described herein. However, in Figure 14 In the integrated sensor ring shown, the meniscus fluid port is entirely formed within the integrated sensor ring, and there is no fluid channel between the microscope objective body and the integrated sensor ring. As previously mentioned, the integrated sensor ring may also include a sensor for immersion control. Therefore, as shown in the figure, Figure 14 The integrated sensor ring may include a supply fluid port in fluid communication with the interior of a meniscus fluid port, which may extend circumferentially around the surface of the integrated sensor ring, partially or entirely, and may surround the microscope objective lens during installation. Furthermore, as explained in detail above, Figure 14 The integrated sensor ring may include sensor interfaces for sensors used for immersion control. Figure 14 The microscope objective body shown may have the same or similar control circuitry system as described above. Figure 13 The integrated microscope objective shown in the diagram has similar electronic and data connectivity features and capabilities.

[0056] Now for reference Figure 15 This describes another embodiment in which the modular sensor ring can be mounted together with the modular microscope objective body. Figure 15 The image depicts the distal portion of the modular microscope objective body. The modular sensor ring and the modular microscope objective body can be formed as separate components or as a single integrated component, and may include internal channels or conduits. Figure 15 The modular sensor ring and modular microscope objective body can be assembled into a similar configuration. Figure 13 The final structure of the integrated microscope objective is shown. However, because... Figure 15The modular sensor ring in the lens can be removable, so it can be easily replaced during use, such as for maintenance, repair, or extending the service life of modular microscope objectives used for immersion microscopy. Figure 15 The modular sensor ring includes an integrated fluid path within the objective lens body, wherein the connection path in the modular sensor ring connects to a sensor plate for detecting the meniscus. As described above, Figure 15 The fluid port at the end passes through with Figure 14 A similar spacing separates the objective lens body. In some embodiments, the spacer ring between the fluid port and the objective lens body may be removed or absent, thereby enabling contact between the fluid and the objective lens body, wherein the modular sensor ring and the objective lens body form a meniscus fluid port in the annular space. In some embodiments, the modular sensor ring and the modular microscope objective lens body can be formed using additive manufacturing techniques such as 3D printing, using any one or more of a variety of suitable materials, including metals and polymers, as well as composite materials. Figure 15 The microscope objective body shown may have the same or similar control circuitry system as described above. Figure 13 The integrated microscope objective shown in the diagram has similar electronic and data connectivity features and capabilities.

[0057] As disclosed herein, the fluid immersion control system can use a common electrode and multiple sensor electrodes at a flat surface associated with the distal end of an immersion microscope objective to monitor the resistance of the fluid as an indication of the presence of a fluid layer having a meniscus diameter larger than the optical axis used for immersion microscopy. When the resistance-indicating diameter is not immersed in the fluid, the fluid immersion control system can activate fluid replenishment.

[0058] The subject matter disclosed above is intended to be illustrative rather than restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments falling within the true spirit and scope of this disclosure. Therefore, to the fullest extent permitted by law, the scope of this disclosure should be determined by the broadest permissible interpretation of the following claims and their equivalents, and should not be limited or construed by the foregoing detailed description.

Claims

1. A control system for fluid immersion in inverted microscopy, the control system comprising: A sensor ring coupled to the distal portion of a microscope lens used with an inverted microscope, wherein the sensor ring forms a band in fluid communication with the microscope lens, wherein the band is capable of delivering fluid that forms an immersion layer on the surface of the microscope lens during imaging using the microscope lens, and wherein the sensor ring further comprises: A plurality of sensor electrodes are circumferentially positioned around the annular band, wherein each of the plurality of sensor electrodes remains in contact with the fluid when the microscope lens is immersed in fluid; A common electrode located within the annular band, wherein the common electrode maintains fluid communication with the fluid; and Fluid port for receiving additional fluid to replenish the immersion layer; A fluid replenishment system, coupled to the fluid port and capable of providing the additional fluid; An immersion controller, coupled to the plurality of sensor electrodes, the common electrode, and the fluid replenishment system, is capable of: When the microscope lens is immersed in fluid, the first resistance of the fluid between each of the plurality of sensor electrodes and the common electrode is measured to detect the fluid at each corresponding sensor electrode. When the fluid is exposed to a first sensor electrode included among the plurality of sensor electrodes, a second resistance of the air gap at the first sensor electrode is measured; and In response to the detection of an air gap, the fluid replenishment system is controlled to provide the additional fluid.

2. The control system as described in claim 1, further comprising: A sensor interface is provided to couple the immersion controller to the plurality of sensor electrodes and the common electrode, wherein the sensor interface further includes: A current source capable of forming a circuit between the common electrode and the plurality of sensor electrodes; and The signal conditioning unit is capable of generating multiple voltage signals from the multiple sensor electrodes respectively.

3. The control system of claim 2, wherein the immersion controller capable of measuring the first resistance and the second resistance further comprises: The immersion controller can be used for: Receive the plurality of voltage signals; as well as Generate multiple digital voltage values ​​corresponding to the multiple voltage signals.

4. The control system of claim 3, wherein the immersion controller further comprises: A multiplexer capable of receiving the multiple voltage signals; as well as An analog-to-digital converter used to generate digital voltage values ​​from the output of the multiplexer.

5. The control system of claim 4, wherein the multiplexer includes a sample-and-hold circuit system.

6. The control system of claim 3, wherein the immersion controller further comprises: Multiple analog-to-digital converters that receive the multiple voltage signals to generate multiple digital voltage values.

7. The control system of claim 1, wherein the fluid replenishment system further comprises: A storage device capable of storing a certain volume of additional fluid; as well as The pump is in fluid communication with the reservoir and the fluid port.

8. The control system of claim 1, wherein controlling the fluid replenishment system to provide the additional fluid further comprises: The additional fluid is supplied until a first resistance is measured from each of the plurality of sensor electrodes, respectively.

9. The control system of claim 1, wherein the microscope lens is located near the transparent sample plate, and wherein the fluid forms a meniscus between the transparent sample plate and the sensor ring surrounded by the distal end of the microscope lens.

10. The control system of claim 9, wherein the inverted microscope is capable of imaging different samples on the transparent sample plate by moving at least one of the transparent sample plate and the microscope lens relative to each other.

11. The control system of claim 10, wherein the transparent sample plate and the microscope lens are movable relative to each other in a plane perpendicular to the optical axis of the microscope lens.

12. The control system of claim 10, wherein the immersion controller is capable of maintaining a first diameter of the meniscus greater than a second diameter of the microscope lens, wherein the second diameter corresponds to the optical axis of the microscope lens.

13. The control system of claim 12, wherein the immersion controller is capable of maintaining the first diameter of the meniscus when the transparent sample plate and the microscope lens move relative to each other.

14. The control system of claim 12, wherein the plurality of sensor electrodes, circumferentially positioned around the annular band, define a first diameter.

15. The control system of claim 1, wherein the sensor ring and the distal portion of the microscope lens form a flat surface, wherein the distal portion of the microscope lens and the plurality of sensor electrodes are at the same height.

16. The control system of claim 1, wherein the plurality of sensor electrodes comprises at least three sensor electrodes.

17. A fluid-immersion microscope objective for inverted microscopy, the microscope objective comprising: A sensor ring coupled to the distal portion of a microscope objective used with an inverted microscope, wherein the sensor ring forms a band in fluid communication with the microscope objective, wherein the band is capable of delivering fluid that forms an immersion layer on the surface of the microscope objective during imaging using the microscope objective, and wherein the sensor ring further comprises: A plurality of sensor electrodes are circumferentially positioned around the annular band, wherein each of the plurality of sensor electrodes remains in contact with the immersion layer when the microscope objective is immersed in fluid; A common electrode located within the annular band, wherein the common electrode maintains fluid communication with the fluid; and Fluid port for receiving additional fluid to replenish the immersion layer; The microscope objective lens described herein can be operated in the following ways: A fluid replenishment system, coupled to the fluid port and capable of providing the additional fluid; An immersion controller, coupled to the plurality of sensor electrodes, the common electrode, and the fluid replenishment system, is capable of: When the microscope objective is immersed in fluid, the first resistance of the fluid between each of the plurality of sensor electrodes and the common electrode is measured to detect the fluid at each corresponding sensor electrode. When the fluid is exposed to a first sensor electrode included among the plurality of sensor electrodes, a second resistance of the air gap at the first sensor electrode is measured; and In response to the detection of an air gap, the fluid replenishment system is controlled to provide the additional fluid.

18. The microscope objective of claim 17, further comprising: A sensor interface is provided that allows the immersion controller to be coupled to the plurality of sensor electrodes and the common electrode.

19. The microscope objective of claim 17, wherein the fluid replenishment system further comprises: A storage device capable of storing a certain volume of additional fluid; as well as The pump is in fluid communication with the reservoir and the fluid port.

20. The microscope objective of claim 17, wherein controlling the fluid replenishment system to provide the additional fluid further comprises: The additional fluid is supplied until a first resistance is measured from each of the plurality of sensor electrodes, respectively.

21. The microscope objective of claim 17, wherein the microscope objective is located near the transparent sample plate, and wherein the fluid forms a meniscus between the transparent sample plate and the sensor ring.

22. The microscope objective of claim 21, wherein the inverted microscope is capable of imaging different samples on the transparent sample plate by moving at least one of the transparent sample plate and the microscope objective relative to each other.

23. The microscope objective of claim 22, wherein the transparent sample plate and the microscope objective are movable relative to each other in a plane perpendicular to the optical axis of the microscope objective.

24. The microscope objective of claim 22, wherein the immersion controller is capable of maintaining a first diameter of the meniscus greater than a second diameter of the microscope objective.

25. The microscope objective of claim 24, wherein the immersion controller is capable of maintaining the first diameter of the meniscus when the transparent sample plate and the microscope objective move relative to each other.

26. The microscope objective of claim 24, wherein the plurality of sensor electrodes positioned circumferentially around the groove define a first diameter.

27. The microscope objective of claim 17, wherein the sensor ring and the distal portion of the microscope objective form a flat surface, wherein the distal portion of the microscope objective and the plurality of sensor electrodes are at the same height.

28. The microscope objective of claim 17, wherein the plurality of sensor electrodes comprises at least three sensor electrodes.

29. A method for inverted immersion microscopy, the method comprising: Using a microscope objective to observe a first sample among a plurality of samples loaded in an inverted microscope, wherein the microscope objective is immersed in a fluid at its distal end, and wherein the microscope objective further comprises: A sensor ring, coupled to the distal portion and forming a band in fluid communication with the microscope objective, wherein the band is capable of delivering fluid, and wherein the sensor ring further comprises: A plurality of sensor electrodes are circumferentially positioned around the annular band, wherein each of the plurality of sensor electrodes remains in contact with the fluid when the microscope objective is immersed in fluid; A common electrode located within the annular band, wherein the common electrode maintains fluid communication with the fluid; and A fluid port for receiving additional fluid from the fluid replenishment system to replenish the fluid; and Using an immersion controller coupled to the plurality of sensor electrodes, the common electrode, and the fluid replenishment system, the fluid is maintained along the diameter of the optical axis of the distal portion when at least one of the microscope objective and the first sample moves relative to each other, wherein maintaining the fluid along the diameter of the optical axis of the distal portion further includes: measuring a first resistance of the fluid between each of the plurality of sensor electrodes and the common electrode when the fluid is immersed in the microscope objective, to detect the fluid at each corresponding sensor electrode of the plurality of sensor electrodes; When the fluid is exposed to a first sensor electrode included among the plurality of sensor electrodes, a second resistance of the air gap at the first sensor electrode is measured; and In response to the detection of an air gap, the fluid replenishment system is controlled to provide the additional fluid.

30. The method of claim 29, further comprising: At least one of the microscope objectives and the first sample is moved relative to each other so as to observe a second sample included in the plurality of samples using the microscope objectives; as well as Maintaining the fluid along the diameter of the optical axis at the distal end also includes maintaining the fluid along the diameter of the optical axis before the microscope objective is aligned with the second sample.

31. A method for inverted immersion microscopy, the method comprising: Using a microscope objective to image a first sample included among multiple samples, wherein the microscope objective is adapted to be immersed in a fluid at its distal end, and wherein the microscope objective further comprises: A sensor ring, coupled to the distal portion and forming a band in fluid communication with the microscope objective, wherein the band is capable of delivering fluid, and wherein the sensor ring further comprises: A plurality of sensor electrodes are circumferentially positioned around the annulus, wherein each of the plurality of sensor electrodes is adapted to remain in contact with the fluid when the fluid immerses the microscope objective. The common electrode located within the annulus is adapted to remain in contact with the fluid when the microscope objective is immersed in the fluid; and A fluid port for receiving additional fluid to replenish the existing fluid; and Using an immersion controller coupled to the plurality of sensor electrodes and the common electrode, the fluid is maintained along the optical axis diameter of the distal portion of the microscope objective as the microscope objective and at least one of the first sample move relative to each other. The method of maintaining the fluid along the diameter of the optical axis at the distal end further includes: when the fluid is immersed in the microscope objective, measuring the first resistance of the fluid between each of the plurality of sensor electrodes and the common electrode to detect the fluid at each corresponding sensor electrode of the plurality of sensor electrodes; When the fluid is exposed to a first sensor electrode included among the plurality of sensor electrodes, a second resistance of the air gap at the first sensor electrode is measured; and In response to the detection of an air gap, fluid is replenished via the fluid port.

32. The method of claim 31, further comprising: A first resistance is detected from a first sensor electrode, wherein the first resistance indicates that fluid is in contact with the first sensor electrode; The fluid supply is stopped in response to the detection of the first resistance from the first sensor electrode.

33. The method of claim 31, further comprising: A circuit is formed between the common electrode and each of the plurality of sensor electrodes by means of a current source; Multiple voltage signals are generated from the multiple sensor electrodes by a signal conditioning unit; Receive the plurality of voltage signals; as well as Generate multiple digital voltage values ​​corresponding to the multiple voltage signals.

34. The method of claim 33, further comprising: The multiple voltage signals are received by an enabled multiplexer; as well as The digital voltage value is generated from the output of the multiplexer by an analog-to-digital converter; The plurality of voltage signals are received by multiple analog-to-digital converters to generate the plurality of digital voltage values; A certain volume of fluid is stored in a storage device; as well as Fluid is pumped from the reservoir to the fluid port via a pump that is in fluid communication with the reservoir.

35. The method of claim 31, wherein the plurality of sensor electrodes are circumferentially positioned around the annular band.

36. The method of claim 31, wherein the plurality of sensor electrodes comprises at least three sensor electrodes.

37. The method of claim 31, wherein the plurality of sensor electrodes comprises at least five sensor electrodes.

38. The method of claim 31, wherein the sensor ring and the distal portion of the microscope objective form a flat surface.

39. The method of claim 38, wherein the distal portion of the microscope objective and the sensor electrode are at the same height.

40. The method of claim 38, wherein the step of imaging a first sample comprising a plurality of samples using a microscope objective further comprises taking an image.

41. A control system for fluid immersion in an inverted microscope, the control system comprising: A sensor ring, coupled to the distal portion of a microscope objective used with an inverted microscope, wherein the sensor ring forms a band in fluid communication with the microscope lens, the band being capable of delivering fluid forming an immersion layer at the surface of the microscope objective during imaging with the microscope objective, and wherein the sensor ring further comprises: A plurality of sensor electrodes are circumferentially positioned around the annulus, wherein each of the plurality of sensor electrodes is adapted to remain in contact with the fluid when the fluid immerses the microscope objective. The common electrode located within the annulus is adapted to remain in contact with the fluid when the microscope objective is immersed in the fluid; and A fluid port for receiving additional fluid to replenish the immersion layer; and An immersion controller, coupled to the plurality of sensor electrodes and the common electrode, is capable of maintaining the fluid along the optical axis diameter of the distal portion of the microscope objective as at least one of the microscope objective and the first sample moves relative to each other, wherein the immersion controller is capable of: When the microscope lens is immersed in fluid, the first resistance of the fluid between each of the plurality of sensor electrodes and the common electrode is measured to detect the fluid at each corresponding sensor electrode. When the fluid is exposed to a first sensor electrode included among the plurality of sensor electrodes, a second resistance of the air gap at the first sensor electrode is measured; and In response to the detection of an air gap, additional fluid is provided via the fluid port to maintain the fluid along the optical axis diameter of the distal portion of the microscope objective.

42. The control system according to claim 41, further comprising: A sensor interface enabling the immersion controller to be coupled to the sensor electrodes and the common electrode, wherein the sensor interface further includes: A current source capable of forming a circuit between the common electrode and the sensor electrode; and The signal conditioning unit is capable of generating multiple voltage signals from the multiple sensor electrodes respectively.

43. The control system of claim 42, wherein the immersion controller further comprises an immersion controller capable of operating as follows: Receive the plurality of voltage signals; and Generate multiple digital voltage values ​​corresponding to the multiple voltage signals.

44. The control system of claim 43, wherein the immersion controller further comprises: A multiplexer is used to receive the multiple voltage signals; as well as An analog-to-digital converter is used to generate the digital voltage value from the output of the multiplexer.

45. The control system of claim 41, wherein the plurality of sensor electrodes are circumferentially positioned around the annular band.

46. ​​The control system of claim 45, wherein the plurality of sensor electrodes comprises at least three sensor electrodes.

47. The control system of claim 45, wherein the plurality of sensor electrodes comprises at least five sensor electrodes.

48. The control system of claim 41, wherein the sensor ring and the distal portion of the microscope objective form a flat surface.

49. The control system of claim 41, wherein the distal portion of the microscope objective and the sensor electrode are at the same height.

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