Self-calibration and directional focusing system and method for infinity corrected microscopes - Patents.com

Abstract

A system and method for auto-focusing an objective lens in a microscope system. The self-calibrating auto-focusing system includes a light source, an eccentric aperture, and an image capture device. The system can be connected to the microscope system such that the light source generates light on an optical path that includes the objective lens and a plate having a reference surface proximal to a sample-holding component. The image capture device images the reflection of the light from the reference surface as the objective lens moves to a series of z-positions. A reference calibration gradient is generated for the objective lens by determining the position of an image to be taken at the z-position of the objective lens. At least one image having specific attributes corresponds to a best focus position. The objective lens is moved from best focus calibrated using the reference calibration gradient to a predicted best focus or a preferred offset.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 16 / 397,665, filed April 29, 2019, the contents of which are incorporated herein by reference in their entirety. FIELD OF THE DISCLOSURE

[0002] The present subject matter relates to systems and methods for focusing a microscope, and more particularly, to systems and methods for calibrating a best focus position for autofocusing a microscope objective. [Background technology]

[0003] Microscopes have evolved from stand-alone arrangements of optics that allow a user to view images from individual slides to high-throughput imaging systems that generate images of samples deposited in multiple sample wells formed in a tray or plate (also known as "microwells," "microtiters," and "microplates"). The optics in a microscope system may be controlled by electromechanical devices under computer program control. Images may be collected using an image capture device, such as, for example, a camera, a charge-coupled device, or other image capture device.

[0004] Modern microscope systems can be used to detect and measure optical signal properties. In some systems, the sample wells may contain materials, such as biological materials, that have been treated with markers or reactive components that cause the emission of light. Microscopes can be used to detect light intensity levels and patterns as opposed to simply capturing images. Thus, microscopes may use signal detector devices and image capture devices to obtain measurements of light levels. The detection components of a microscope may thus include, for example, photomultipliers, photodetectors, and photodetector arrays in addition to image capture devices. Microscope systems may also provide alternative illumination systems, such as, for example, coherent (laser) or non-coherent light sources, or light sources filtered at selected wavelengths. Filters, beam splitters, and other optical devices may be inserted at various locations in the optical path between the light source and the sample, or between the sample and the detection components, to either excite the sample in a desired manner or measure selected optical properties, including intensity and other properties at certain wavelengths.

[0005] In capturing or determining a pattern in an image or measuring an optical signal, optical devices such as lenses, mirrors, filters, etc. are placed along the optical path from the light source to the image capture device. The imaging lens is adjusted to focus the light source onto the image plane of the image capture device as the objective lens focuses on the reference surface. Lack of focus appears as blurring or blurring in the image. Similarly, optical measurements obtained from an unfocused objective lens may not yield accurate intensity levels and pixels that may be brighter or less bright than expected due to the spread of the image when defocused. The objective lens should typically be positioned to focus on the sample before imaging or optical measurements are performed by the microscope system.

[0006] In a high-throughput microscope system, where the microscope may be used to capture images or optical signal measurements of a large number of samples, the focusing step may need to be performed as quickly and as many times as possible. The focusing process should be sensitive and accurate, while adding as little complexity as possible and perturbing the sample. The focusing process should also be an automatic focusing process, or a process that is performed with as little user involvement as possible. Fluorescence microscopes are used in analyzing biological samples that are labeled with one or more fluorophores. It is important to avoid causing optical damage to the fluorophores and the biological sample.

[0007] Microscope systems typically perform hardware-based autofocusing on each sample being processed. In high-throughput microscope systems, sample wells supported on a plate have a sample well bottom surface and a plate bottom surface. The sample well bottom surface and the plate bottom surface are at least partially reflective. In one approach to a hardware-based autofocusing system, the sample wells are illuminated through an objective lens to be used in the imaging or optical measurement to be performed on the sample in the sample well. The objective lens is positioned at a series of positions along an optical axis along which the objective lens travels, while the image and pixel intensity are checked at each position. The optical axis along which the objective lens travels may be referred to as the "z-axis," and the motion of the objective lens may be referred to as "scanning along the z-axis."

[0008] FIG. 1A is a schematic diagram of a microscope 100 illustrating the operation of a process for implementing hardware-based autofocusing of the microscope 100 to image a sample 114 in a sample well 112 disposed on a plate 110. The microscope 100 in FIG. 1A includes the main components for autofocusing: an objective lens 102, a detector 104, a beam splitter 106, and a light source 108. The microscope 100 may be implemented in a system including components configured to control the state and motion of the objective lens 102 and the plate 110. Such components may also be configured to control the state of the light source 108 and the operation of the detector 104. For example, the movement of the objective lens 102 along the z-axis may be controlled using a linear actuator having an electric motor under the control of a computer program. The objective lens 102 is moved to a position on the z-axis (i.e., a z-position). The detector 104 may be implemented as a charge-coupled device or other image capture device controlled by a computer program to capture an image and transfer the digital content of the image to a computer.

[0009] In an exemplary hardware-based autofocus process, a user may be provided with a user interface for inputting configuration data to configure the microscope according to the type of objective 102, the type of plate 110, and the light source 108. The system may then position the objective 102 at a starting location on the z-axis that is suitably close to the plate 110. The starting location may depend on the type of plate 110, as different plates may have plate bottom surfaces at different locations along the z-axis. The objective 102 may be controlled to move along a working distance or scanning range where the best focus position may be found. This process typically involves positioning the objective 102 at each z-position within the scanning range and capturing an image at each z-position.

[0010] Light from the light source 108 follows a first optical path at 101 from the light source 108 to the beam splitter 106. The beam splitter 106 reflects a portion of the light towards the objective lens 102, which directs the light along optical path 105 towards the sample well 112. The light reflects back from the top and / or bottom surfaces of the plate 110 towards the objective lens 102, through the beam splitter 106, along optical path 107 to the detector 104. If the position of the objective lens 102 is close enough to where it focuses onto the reflective surface of the plate 110, the light will appear at the detector 104 as a beam spot with a certain diameter and a certain intensity. As the objective lens 102 is moved along the z-axis, the system controls the detector 104 to capture an image at each z-axis position. Each image is analyzed to determine a focus score based on focus metrics that may include, for example, the average intensity, maximum signal, standard deviation, variance, or covariance, size, morphology, location, and others, of the pixels of each spot. The smallest and brightest spot is typically considered to have the "best focus." The focus score may be plotted in a graph 120 versus the z-position at which each image is captured. In the graph 120 (see FIG. 1B), the smallest and brightest spots may form a peak at 124 in terms of their intensity and z-position value. The graph 120 may show the brightest pixels as peaks 122 and 126. The z-position at the highest focus score (at 124 in FIG. 1B) is identified as being the best focus position.

[0011] Note that two peaks may be formed in the graph, indicating reflection from two surfaces on plate 110. Once a peak or peaks have been identified and a corresponding z-position identified for each peak, the process identifies the best focus position as the z-position corresponding to the selected one of the peaks. That is, the best focus position is the position where the spot formed by light reflected from either the top or bottom surface of plate 110 has the best focus score.

[0012] The best focus position in the context of the autofocus procedure described with reference to FIG. 1A is the z-position of the objective lens 102 that produces the sharpest and clearest projection of the light beam reflected from the reference surface. In the system shown in FIG. 1A, the reference surface is either the top or bottom surface of the plate 110. For example, to image with good focus on a sample (114 in FIG. 1A), such as a collection of cells, the objective lens 102 should be moved to focus on the sample 114. In some implementations, an offset along the z-axis may be defined according to the sample type, the objective lens, the plate type, and / or other suitable parameters. Once the best focus position is determined, the objective lens 102 may be moved according to the offset and begin imaging. In some implementations, the system may perform image-based autofocusing after hardware-based autofocusing. Image-based focusing involves moving the objective lens 102 along the z-axis while checking the actual image of the sample until the sample is in focus.

[0013] The autofocusing procedure described with reference to FIG. 1A may involve hardware-based autofocusing to find a reference surface on the plate 110 and either moving with an offset to focus on the sample or image-based focusing to find the sample 114. Hardware-based autofocusing, image-based autofocusing, or both may need to be performed each time a new sample well is provided for imaging, even if the objective lens is not changed. The microplate in which the sample wells are located may not be perfectly flat, and the thickness of the sample well bottom may not be consistent. Different samples or different light sources may also require refocusing.

[0014] Current hardware-based autofocusing procedures are slowed down by the need to collect a data point from each z-position within the working distance of the objective 102. In systems where the microscope is used to image multiple samples, even a short delay in focusing the microscope objective may be too long. Other processes used involve trade-offs and other limitations, such as complexity, reduced accuracy, sensitivity to surfaces that reflect the imaged beam during focusing, high cost, etc. Summary of the Invention [Means for solving the problem]

[0015] According to one aspect, a method is provided for performing a self-calibrating autofocus procedure for an objective lens in a microscope system. In an exemplary method, a reference calibration gradient is generated by determining the position of each of a plurality of images taken at a series of z-positions of the objective lens. The images are analyzed to determine a particular attribute that corresponds to a best focus position. An autofocus procedure can then be performed on the objective lens based at least in part on the reference calibration gradient and the best focus position.

[0016] In another aspect, the reference calibration gradient describes a linear relationship between a set of objective lens positions and lateral positions on the image plane as the reference image is translated within the image plane.

[0017] In another aspect, the reference image can be a beam spot reflected from a reference surface and projected onto an image capture device. Alternatively, the reference image can be a patterned image.

[0018] In another aspect, a system for auto-focusing is provided. One implementation of the auto-focusing system includes a light source, an off-center aperture, and optical components for forming an optical path toward an image capture device. The auto-focusing system may be connected to a microscope system with an optical path including an objective lens of the microscope system. The auto-focusing system may also operate as a subsystem of the microscope system. The auto-focusing system may also include a controller or operate using a controller operating within the microscope system. The controller may include storage for machine instructions that, when executed by a processor, implement a self-calibrating auto-focusing method. The present specification also provides, for example, the following items: (Item 1) 1. A method of automatic focusing for an objective lens in a microscope, the method comprising: generating a reference calibration gradient for the objective lens by determining respective positions of a plurality of images taken at a series of z-positions of the objective lens, where at least one image of the plurality of images has a particular attribute corresponding to a best focus position, and an optical path formed during capture of the plurality of images is at least partially blocked by an off-center aperture; autofocusing the objective lens based at least in part on the reference calibration gradient and the best focus position; A method comprising: (Item 2) Item 10. The method of item 1, wherein the multiple images comprise a beam spot in each image formed by projecting light reflected from a reference surface onto an image capture device. (Item 3) Item 10. The method of item 1, wherein the plurality of images comprises a pattern in each image formed by projecting patterned light reflected from a reference surface onto an image capture device. (Item 4) 4. The method of claim 3, wherein the particular attribute corresponding to the best focus position is a minimum pattern image size or an optimal contrast. (Item 5) 4. The method of claim 3, wherein the particular attribute corresponding to the best focus position is the highest pattern correlation with an original pattern using pattern recognition techniques. (Item 6) 2. The method of claim 1, wherein the particular attribute corresponding to the best focus position is highest contrast. (Item 7) 2. The method of claim 1, wherein the particular attribute corresponding to the best focus position is a minimum beam spot size. (Item 8) 2. The method of claim 1, wherein the particular attribute corresponding to the best focus position is maximum illumination. (Item 9) 2. The method of claim 1, wherein determining the positions of the plurality of images includes determining a center of mass location of each image along an axis of lateral translation. (Item 10) 2. The method of claim 1, wherein generating the reference calibration gradient includes determining a linear relationship between the center of mass locations of the images and the corresponding z positions. (Item 11) positioning another sample well in the optical path of the objective; identifying a distance to move the objective lens to the best focus position for the other sample well; Further comprising: The identifying step comprises: Capturing another reference image; identifying the lateral location on an image plane of the other reference image; Calculating a distance to the best focus position based on the lateral position of the other reference image and the reference calibration gradient; The method according to item 9, (Item 12) positioning the objective lens at the best focus position; determining an offset from the best focus position to a sample focus position; positioning said objective lens at said best focus position plus said offset; The method of claim 1, further comprising: (Item 13) moving the objective lens to the best focus position; capturing an image of the sample at each of a plurality of z positions in a direction toward the sample; determining a focus score for each sample image at each corresponding z-position based on any one or combination of focus metrics including spot diameter, intensity, mean intensity, maximum signal measure, standard deviation, variance, and covariance; plotting said focus scores for each sample image for each z position; Identifying the highest peak in the final score; positioning the objective lens at the z position corresponding to the highest peak; The method of claim 1, further comprising: (Item 14) 1. A system for auto-focusing an objective lens in a microscope system, the system comprising: A light source; an image capture device configured to capture images of light reflection from a reference surface while the objective lens is moved through a series of z positions along a z axis; and an off-center aperture in an optical path from the light source, the off-center aperture blocking at least a portion of the light from the light source; a controller configured to control the light source, the image capture device, and the objective lens; Equipped with The controller is configured to perform a self-calibrating auto-focusing process, and performing the self-calibrating auto-focusing process includes: generating a reference calibration gradient for the objective lens by determining respective positions of a plurality of images taken at a series of z-positions of the objective lens, at least one image of the plurality of images having a particular attribute corresponding to a best focus position; autofocusing the objective lens based at least in part on the reference calibration gradient and the best focus position; By,System. (Item 15) Item 15. The system of item 14, wherein the light source is configured to project a beam spot onto the image capture device. (Item 16) Item 15. The system of item 14, wherein the light source is configured to project a pattern image onto the image capture device. (Item 17) Item 17. The system of item 16, wherein the particular attribute corresponding to best focus is a minimum pattern image size or an optimal contrast. (Item 18) Item 17. The system of item 16, wherein the particular attribute corresponding to the best focus position is the highest pattern correlation with an original pattern using pattern recognition techniques. (Item 19) Item 15. The system of item 14, wherein the particular attribute corresponding to the best focus position is highest contrast. (Item 20) Item 15. The system of item 14, wherein the particular attribute corresponding to the best focus position is a minimum beam spot size. (Item 21) Item 15. The system of item 14, wherein the particular attribute corresponding to the best focus position is highest illumination. [Brief description of the drawings]

[0019] The drawings illustrate the design and utility of embodiments of the disclosed invention, where similar elements are referenced by common reference numbers. The drawings are not necessarily drawn to scale. The drawings depict only exemplary embodiments of the disclosed invention, and therefore should not be considered as limiting its scope.

[0020] [Figure 1A] FIG. 1A is an example of a known microscope system configured to implement a hardware or image-based autofocus procedure.

[0021] [Figure 1B] FIG. 1B is an example of a graph of focus scores related to intensity measurements of light reflected from two surfaces in a sample well for a scan of the z-position relative to the objective lens.

[0022] [Figure 2A] FIG. 2A is an example implementation of a system for calibrating the best focus position and auto-focusing an objective lens in a microscope system.

[0023] [Figure 2B] FIG. 2B is a flow chart illustrating the operation of an exemplary system for calibrating a best focus position and auto-focusing an objective lens in a microscope system.

[0024] [Figure 3A]FIG. 3A is a schematic diagram of an example of multiple images illustrating lateral translation of a beam spot overlaid on a representation of a linear array detector during calibration of a best focus position for an objective lens in a microscope system.

[0025] [Figure 3B] FIG. 3B is an exemplary centroid plot of the laterally translated beam spot location on the detector during a scan of the z position of the objective lens.

[0026] [Figure 4-1] 4A-4C are schematic diagrams of example images captured at different z-positions of an objective lens illustrating the process for auto-focusing when a reference calibration gradient is available for the objective lens.

[0027] [Figure 4-2] FIG. 4D is an example of a patterned image that can be projected onto an image capture device as a reference image.

[0028] [Diagram 5] FIG. 5 is another example of a microscope system configured to perform self-calibrating autofocusing with an off-center aperture 502 in the collimated illumination path.

[0029] [Figure 6] FIG. 6 is another example of a microscope system configured to perform self-calibrating autofocusing with an off-center aperture 602 in the collimated return emission path on the detector side.

[0030] [Figure 7] FIG. 7 is another example of a microscope system configured to perform self-calibrating autofocus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] All numerical values ​​are assumed herein to be modified by the term "about" or "approximately", whether or not expressly indicated, and the terms "about" and "approximately" generally refer to a range of numbers that a person skilled in the art would consider equivalent to the recited value (i.e., having the same function or result). In some cases, the terms "about" and "approximately" may include numbers that are rounded to the nearest significant figure. Recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or," unless the content clearly dictates otherwise. In describing the depicted embodiments of the disclosed invention, which are illustrated in the accompanying figures, specific terminology has been employed for clarity and ease of description. However, it is to be understood that the disclosure of this patent specification is not intended to be limited to the specific terminology so selected, and that each specific element includes all technical equivalents that operate in a similar manner. Furthermore, it is to be understood that various elements and / or features of different illustrative embodiments may be combined with each other and / or substituted for each other, where possible within the scope of this disclosure and the appended claims.

[0033] Various embodiments of the disclosed invention are described below with reference to the figures. Please note that the figures are not drawn to scale, and that elements of similar structure or function are represented by similar reference numbers throughout the figures. Please also note that the figures are intended to facilitate the description of the embodiments only. They are not intended as a comprehensive description of the invention, or as a limitation on the scope of the disclosed invention, which is defined solely by the appended claims and their equivalents. In addition, an illustrated embodiment of the disclosed invention need not have all of the aspects or advantages shown. For example, an aspect or advantage described in conjunction with a particular embodiment of the disclosed invention is not necessarily limited to that embodiment, and can be practiced in any other embodiment, even if not so illustrated.

[0034] A system and method for determining a best focus position for an objective lens in a microscope system is described below. The method and system described below disclose a calibration process that generates a reference gradient corresponding to various known parameters. Unique to the inventive concept, an off-center aperture is utilized to provide sampling of the wavefront at multiple z-positions that are strategically mapped to form a calibration reference gradient. This reference gradient derived through the calibration process can then be advantageously used to determine an offset from the best focus position, as will be discussed in more detail below. This approach allows the system to quickly calibrate and return to best focus during image acquisition, minimizing errors and improving throughput.

[0035] As used in the following description, the term "best focus position" shall mean the z-position of the objective lens at which the objective lens provides the best focus on the reference image projected onto the reference surface. Thus, the best focus position may not be the position at which the objective lens focuses on the sample. The reference surface used to determine the best focus position may be below or above the z-position level of the sample when the sample is presented for imaging. In some example implementations, and for certain imaging or optical measurement procedures, an offset from the best focus position may be defined based on the type of sample being imaged and the objective lens being used for imaging. When more precision is required, hardware focusing may be performed similar to that described above with reference to Figures 1A and 1B. The advantage to starting such hardware-based focusing from a location close to the best focus position is that fewer z-positions are used than with the procedure described above that does not start from the best focus position.

[0036] As used in the following description, the terms "imaging," "image capture," "capturing an image," or "detecting an image" shall refer to any process for collecting optical data from an image capture device. The image may be a reference image for acquiring a reference calibration gradient, an image of a sample, including capturing a digital image for storage, or measuring optical properties such as intensity, color, or other types of data.

[0037] As used in the following description, the term "sample well" refers to any suitable structure for containing a sample to be presented for imaging or for obtaining optical measurements. A sample well may include, for example, a recessed structure formed on a microwell plate as defined below into which a sample may be deposited. A sample well, as that term is defined herein, may also include a slide with or without a cover or any other suitable structure for containing a sample.

[0038] As used in the following description, the term "plate" shall refer to any sample-holding structure configured to support a container in which a sample can be deposited. In particular, the term "plate" may include trays or similar structures that include such sample-holding structures, known in the art by terms including "microwell", "consumable", "microtiter" and "microplate". "Plate" shall be understood to refer to a structure capable of holding a single sample well or multiple sample wells. In the examples described below, the plate is used to provide a reference surface for determining the best focus position. The term "plate", unless otherwise stated, shall be understood in the following description to mean any structure that can provide a reference surface for the processes described below.

[0039] As briefly discussed above, the present invention discloses a system and method for calibrating a best focus position for an objective lens in a microscope system and then using the best focus position calibration to focus the objective lens more quickly and efficiently than using conventional autofocusing techniques. When a sample is positioned for measurement using a calibrated objective lens, and after the objective lens is repositioned, a best focus position calibration is used to find the position of the objective lens relative to its best focus position. In order to focus on the sample, the objective lens can be moved to the best focus position or can be moved a predetermined offset from best focus.

[0040] Calibrating the best focus position for a given objective involves determining the relationship between the objective's position on its axis of travel and the translation of an image (or reference image) on the image plane of an image capture device. An autofocusing subsystem or module in a microscope system may include a suitable light source, an off-center aperture, a reference surface, imaging optics, and an image capture device. The reference surface may be a plate (i.e., a microplate, a microwell plate, a microtiter, etc.) that may be used to hold a sample well containing a sample on which optical measurements will be performed. The components may be positioned to illuminate the reference surface through the objective of the microscope system with the light source. The optical components project the reference image onto the image capture device, for example, as shown in Figures 2A and 5-7. The off-center aperture is positioned and sized to sample a portion of the total light through the entrance pupil of the objective and project it, for example, as a beam spot onto the image capture device. As the objective is translated along its axis of travel, the beam spot translates linearly and changes focus. This pattern is strategically utilized to generate a reference gradient, as will be discussed in more detail below, which maps the position of the beam spot to multiple z-positions.

[0041] 2A is an example implementation of an autofocusing system 200 for calibrating a best focus position and autofocusing an objective lens 202 for a microscope system. The autofocusing system 200 includes an image capture device 204, a light source 206, a first imaging lens 208, a beam splitter 210, a second imaging lens 240, and an autofocus aperture 230. The objective lens 202 is a component of the microscope system configured to perform imaging and / or optical measurements on a sample that may be deposited in a sample well 214. Other components of the microscope system include an optional sample holding structure that supports a plate 212 with reference surfaces 212a, 212b.

[0042] The autofocusing system 200 of FIG. 2A is implemented as a module or subsystem of a microscope system. Other components that the microscope system uses to image a sample, such as, for example, an excitation light source, a filter, a beam splitter, and a sample image capture device, are represented in FIG. 2A as a sample imaging component 221. The sample imaging component 221 may include, for example, a lens, a filter, or other optical device that forms an optical path that includes the objective lens 202 and the plate 212 when the microscope system is used to image a sample. The microscope system 200 may also use a different light source or a different sample image capture device based on the type of imaging or measurement being performed. The optical device may be inserted at 221a below the objective lens of FIG. 2A, above the beam splitter 210, or at 221b above the off-center aperture 230. The sample well 214 in the example shown in FIG. 2A is formed on the plate 212, which provides a bottom reference surface 212a and a top reference surface 212b for the autofocusing procedure.

[0043] Once self-calibration of the best focus position has been performed for a given objective lens 202, the process of focusing that objective lens 202 in subsequent imaging or optical measurements can be performed with minimal further imaging. The calibration of the best focus position can be stored as a reference calibration gradient, which can be stored or included with data characterizing the objective lens 202 in the system data storage system 223.

[0044] The first imaging lens 208 collimates the light from the light source 206 along optical path 201 and passes the collimated light to the beam splitter 210. The beam splitter 210 reflects a portion of the light along optical path 203 toward the objective lens 202 and toward a plate 212 on optical path 205. The plate 212 reflects the light toward the beam splitter 210 on optical path 207 back to the objective lens 202. The beam splitter 210 passes a portion of the light along optical path 209 toward an off-center aperture 230. The light passes through an off-center opening on the aperture 230 that is smaller than the entire light beam impinging on the aperture 230. The remaining portion of the aperture 230 blocks the portion of the light beam that is not passed through the aperture. The light that passes through the aperture 230 is directed to the second imaging lens 240 and the image capture device 204. The off-center aperture 230 operates by sampling a portion of the plane wavefront from the objective lens 202. The sampled portion of the light is focused and directed toward the detector by the second imaging lens 240, but is constrained by the off-center aperture as an asymmetric marginal ray. This allows the light and the location of best focus to be viewed without changing any of the component settings. It should be understood that different off-center apertures with different sizes and / or positions can be used to allow for adjustments in sensitivity or different size pupil diameters of the objective lens 202.

[0045] 2A show only the light along the optical paths that form the light beam that impinges on image capture device 204. The portions of light that are not shown are the portions of light that are blocked by the blocking portion of autofocus aperture 230.

[0046] The objective lens 202 is configured to move along optical paths 203 and 205 on the z-axis, which is perpendicular to the xy plane in which the plate 212 extends (shown in FIG. 2A). The following description will refer to the position of the objective lens as being on the z-axis and the position, or lateral position, of the reference image as being on the xy plane, as a way of providing clarity. The reference image translates along a linear path on the image plane. This linear path is referred to as the "y-axis." It should be understood that the use of "z-axis" or "y-axis" to provide spatial designation is not intended to be limiting. Any suitable coordinate system may be used. Additionally, it should be noted that example implementations may involve the objective lens 202 traveling in a non-vertical direction.

[0047] The plate 212 may include a sample well 214, as shown in FIG. 2A, which may be positioned for imaging a sample that may be deposited therein according to the normal function and operation of a microscope system. In the exemplary system shown in FIG. 2A, the plate 212 has a first surface 212a, which is the bottom surface of the sample well, and a second surface 212b. The first surface 212a and / or the second surface 212b may be at least partially reflective, thereby providing a reflective reference surface for use during a self-calibrating autofocus procedure. The reference surface may also be provided on a cover glass, or on a surface of a slide or other planar material, disposed in the optical path proximate to the bottom surface of the sample well 214.

[0048] The objective lens 202 may be moved along the z-axis using a linear actuation motor controlled by the controller 220. The objective lens 202 is diagrammatically represented in FIG. 2A as including a linear actuation motor that moves the objective lens 202. The objective lens 202 includes selected optics configured to focus light from a light source onto a sample 216 when the microscope system 200 is controlled to image on the sample. During an autofocus procedure, the objective lens 202 is controlled to focus on a reference surface (212a or 212b on the plate 212). In some implementations, the motor that moves the objective lens 202 may be a stepper motor or a servo motor with a linear actuator.

[0049] Light along the optical path passing through the off-center aperture 230 travels through the second imaging lens 240 to the image capture device 204, where the projected source is imaged on the detector plane. With defocus, the light beam on the image capture device 204 spreads in size and has lower intensity and / or lower contrast. When the objective lens 202 is in focus, the reference image is captured at maximum intensity, minimum size, and its highest contrast. The process of focusing the objective lens 202 involves moving the objective lens 202 to find the best focus position on the z-axis. Each beam spot in each reference image captured at each objective lens 202 z position appears at a position in the image plane that is offset from the spot position on the previous image.

[0050] In an exemplary implementation, the image capture device 204 in the auto-focusing system 200 may be a linear array detector, a charge-coupled device, a position sensitive diode, a 2D sensor array such as an image capture device, or any suitable device that can be controlled by the controller 220 to capture images of the reference image as the objective lens 202 is controlled to move to a series of z-positions. The light source 206 in the auto-focusing system 200 may be any suitable light emitting device, such as a laser, a light emitting diode (LED) or an LED array, white light, a fluorescent light source, infrared light, etc.

[0051] The controller 220 may be implemented using any computer programmable system having a hardware interface connected to at least the image capture device 204 and a motor configured to move the objective lens 202. In some implementations, the controller 220 may also be a component of a microscope system 221 in which the objective lens is auto-focused. The self-calibrating auto-focusing procedure may be a function stored as software in a data storage medium 223 that the controller 220 has access to.

[0052] The self-calibration and auto-focus procedure for a given objective lens involves performing a first process of obtaining a reference calibration gradient. The reference calibration gradient may then subsequently be used to auto-focus the objective lens. The flowchart of FIG. 2B illustrates the operation of an example method 250 for auto-focusing an objective lens by first obtaining a reference calibration gradient at 260 and using the reference calibration gradient to determine a best focus position of the objective lens 202 at 270. The objective lens 202 may then be moved to focus on the sample using an offset from the best focus position or using other methods such as performing hardware-based focusing on the sample similar to the hardware-based method described above with reference to FIG. 1A.

[0053] 2A and 2B (with respect to components referenced below), a self-calibration and auto-focus procedure may be initiated in step 252 when the objective lens 202 is inserted for use within the microscope system 200. In step 254, the system may perform a start or set function to use the particular objective lens 202 that has been selected. One start step may include determining whether a reference calibration gradient has been generated for the objective lens 202 in decision block 256. If a reference calibration gradient has not been generated (indicated by the "No" path to method 260), a starting point location is determined for the auto-focus procedure in step 262.

[0054] When the objective lens 202 is inserted or installed in a microscope system, the position of the objective lens 202 along the z-axis is typically not known to the system. The system 200 may perform a home function that positions the objective lens 202 at a known position based on a home structure that may be sensed as it moves by a sensor attached to the objective lens 202 or a structure attached to the objective lens 202. In other implementations, the objective lens 202 may be positioned at its furthest location from the plate 212.

[0055] Once the objective lens 202 is in a known or home position, the controller 220 may position the objective lens 202 a predetermined distance away from the home position to begin the autofocus procedure in step 263. The predetermined distance may be a starting position stored in the data description of the objective lens 202. The starting position may be a parameter used for all or most of the objective lens.

[0056] In step 264, a reference image is captured at the current position of the objective lens 202. The objective lens 202 is then positioned at a series of z positions, scanning the reference surface as the objective lens is moved. The scan involves capturing a reference image at each z position. The z positions are checked to determine if the scan is complete, as shown in decision block 265. An example of FIG. 2B for the check used in decision block 265 determines if the scan is complete by checking if a reference image has been captured for each z position of the scan. The scan may be performed with a set number of z positions, or until a travel limit on the objective lens is reached. If the scan is not complete, the objective lens 202 is moved to the next z position in step 266 (along the "no" decision path). In step 264, a reference image is captured at the next z position. If the scan is complete, then in step 268 (the "yes" path of decision block 265), the reference images captured at each z position are analyzed.

[0057] FIG. 3A is a schematic diagram of an example of multiple images 300 illustrating lateral translation (along the y-axis) of beam spots 302a-o overlaid on a representation of a linear array detector 304 during calibration of a best focus position for an objective lens in a microscope system.

[0058] The beam spots 302a-o in Figure 3A are shown at 16 locations on the image plane of the linear array detector 304 for images captured at 16 corresponding z positions of the objective lens 202 (Figure 2A). Note that while Figure 3A shows 16 locations to illustrate this process, a single image plane is acquired per z position each time. It should be understood that while the illustrated embodiment shows 16 beam spots, any number of z positions and corresponding beam spots may be used as well.

[0059] The beam spots 302 in each image are analyzed to determine the location of each beam spot. Determining the center of mass location of an imaged object is well known and need not be described in further detail. The y-axis location of the center of mass of each beam spot 302 in each reference image is determined and related to the z-position of the objective lens when each image was captured. Figure 3A illustrates the lateral translation of the beam spots 302 as the reference images are overlaid on top of each other. The beam spots 302 are located along the y-axis along which they appeared in the reference image from which they were captured.

[0060] Each beam spot 302a-o is shown in FIG. 3A as being unfocused at an initial y-axis location, such as y=1 through y=6. The level of focusing is determined from measuring attributes of the beam spot 302. Beam spots 302a-e have lower intensity, which is shown in the example of FIG. 3A by, for example, the lighter shading of each spot. At y=7, beam spot 302f is more focused, which is determined by the smaller size and higher intensity of beam spot image 302f. The higher intensity is shown by the darkening shading of beam spot 302f. Beam spot 302h is the darkest (highest light intensity) and smallest spot in FIG. 3A.

[0061] As the objective lens 202 is moved further along the series of z positions in the scan beyond y=9, the beam spots 302i-302o become larger and have a lighter shade indicating that the reference image is out of focus. With reference to FIG. 2B, the best focus position may be determined by identifying the smallest and brightest beam spot and setting the z position at which the image was captured as the best focus position, as shown in step 268. In the example shown in FIG. 3A, the best focus position is the z position corresponding to the y position of the beam spot 302h. As shown above, other optical characteristics may also be used to determine the best focus position other than intensity, and the above example should not be read as limiting. In addition, the determination of intensity for each beam spot 302 (of FIG. 3A) may be based on the average intensity or other measurements of all pixels forming the spot. In an exemplary implementation, intensity or contrast may be used to determine a focus score for each spot.

[0062] Further analysis compares the y position of the beam spot (302a-o in FIG. 3A) with the z position to determine a linear relationship between the y and z positions in step 268. The linear relationship may then be stored as a reference calibration gradient in step 269.

[0063] Although plotting data is not necessary to store a reference calibration gradient, FIG. 3B illustrates one example of a plot of reference image position y versus z objective lens position for plate 212a. For convenience, the y and x axes have been shifted relative to the best focus position at the origin. As shown in FIG. 3B, the yz points identified from the scan described with reference to FIG. 3A may lie on a curve C that may be approximated by a line L. The reference calibration gradient for objective lens 202 may be shown as being a linear equation for line L (i.e., y=mz+B), which in the illustrated example is y=1.37z+8.814. For objective lens 202, a reference image such as beam spot 302 may be captured wherever objective lens 202 is positioned. The position of beam spot 302 on the y axis may be determined. The current distance from the best focus position is then determined using y=1.37z+8.814, which can be resolved in z from one or more reference images within the linear range of the system.

[0064] The reference calibration gradient is preferably a non-zero gradient m. A reference calibration gradient of m=0 would result if no off-center aperture or imaging lens were used in the autofocus system. A flat horizontal line would not allow the determination of the best focus position, since the beam spot would be at the same position in all reference images, regardless of the position of the objective lens. The reference calibration gradient can be adjusted by selecting the off-center aperture according to the aperture size and the location of the light passing through a portion of the aperture. An imaging lens can also be added to the autofocus system before or after the off-center aperture to adjust the magnitude of the reference calibration gradient. It should also be noted that the alignment of the sensor to the locus of reference image positions on the sensor can be easily set by the orientation of the off-center aperture relative to the sensor.

[0065] Referring back to FIG. 2B, in decision block 256, the objective lens inserted in step 252 may already have been calibrated for the best focus position. If so, a reference calibration gradient is found for the objective lens 202 (the "Yes" decision path is followed) and method 270 is performed to obtain focus. The reference calibration gradient for the objective lens 202 is retrieved in step 272 and moved to a start position in step 276. In step 278, an image is captured at the start position. In step 280, the image is analyzed to determine the y-axis position of the center of mass of the beam spot in the image. The y-position of the image is then used in step 282 in the reference calibration gradient to determine the current distance between the current position of the objective lens 202 and the best focus position.

[0066] The sample imaging component 221 (of FIG. 2A) of the microscope system may now be deployed to image the sample using the objective lens 202. The objective lens 202 may be moved to focus on the sample by moving the best focus position and a predetermined offset along the z-axis in step 284. The predetermined offset may be determined according to the type of sample being imaged or the plate being used to hold the sample. In some implementations, the offset may depend on the objective lens, the sample, or a longitudinal chromatic offset. The objective lens may be controlled to move to the predetermined offset by shifting the target reference image away from best focus and scaling the shift with the calibration gradient.

[0067] Some optical measurements may require high sensitivity to focus deviations. As described above, the objective lens may be focused on the sample using hardware-based focusing as described above with reference to Figures 1A and 1B, but starting from a best focus position determined using method 250 of Figure 2B. Combining the profile method described with reference to Figures 1A and 1B with the predictive nature of the off-center aperture and calibration gradient will improve the speed of the method by defining a smaller search range. The smaller search range may be based on one or more measurements that predict the appropriate objective lens position, but maximizes sensitivity to focus deviations.

[0068] Referring back to FIG. 2B, if a more precise distance to the best focus position is required, the objective lens can be moved to several different z positions and steps 278-282 can be repeated for each z position to which the objective lens 202 is moved. In this way, not only is the linear relationship that defines the reference calibration slope confirmed, but the best focus can be found based on focus metrics around the best focus by fitting, interpolating, or otherwise to improve resolution to focus deviations. This process is illustrated with reference to FIGS. 4A-4C. FIGS. 4A-4C show graphical representations 400, 402, 406 of images of a beam spot 420 captured with an objective lens at different z positions using the example method 270 of FIG. 2B. Each spot 420 in FIGS. 4A-4C is shown with a center of mass 421 as a smaller spot approximately at the center of the spot. Image 402 in FIG. 4B shows the beam spot 420 at or near the best focus position. Image 400 of FIG. 4A shows beam spot 420 at a y position indicating that objective lens 202 is defocused in one direction away from best focus. Image 406 of FIG. 4C shows beam spot 420 at a y position indicating that objective lens 202 is defocused in the opposite direction of beam spot 420 of FIG. 4A away from best focus. Images of three beam spots are shown in FIGS. 4A-4C for illustrative purposes. The three beam spots may actually represent a scan of a region of interest (ROI) that may be smaller than the image sensor centered around the calibrated best focus, clipping the entire dynamic range of measurable defocus of the objective lens. In an exemplary implementation, many additional images of the beam spot may be captured and analyzed to improve resolution, especially for the smaller ROIs.

[0069] The beam spot 420 of Figures 4A-4C may be analyzed to determine a best focus position and a calibration gradient based on a scan of the three beam spots. The best focus position may be found by interpolating, fitting, or taking the peak focus metrics from the three beam spot scans and then comparing to the original best focus position. The calibration gradient found by analyzing the three beam spot scans may also be compared to the original reference calibration gradient. The best focus position and the reference calibration gradient should be within an acceptable error. If either is not within an acceptable error, the scan may be repeated with more data points or with a larger ROI.

[0070] It should be noted that the reference image can be a beam spot or a patterned image. The light source 206 can be configured to generate white light or any other light of a suitable wavelength as a simple light beam or as a patterned image. FIG. 4D is an example of a patterned image that can be projected onto the reference surface 212a or 212b and detected by an image capture device as a reference image. When a patterned image is used, the best focus can be determined by analyzing the reference image using a suitable data transformation, such as a Fourier transform, and then calculating the correlation. The patterned image can then be analyzed for optical attributes corresponding to best focus, similar to the techniques described above for the beam spot. The lateral position can be determined as either the center of mass or the brightest pixel, and the highest contrast can be a preferred image attribute for determining best focus. The patterned image can also be analyzed using pattern recognition methods, and the best pattern match to the original pattern indicates best focus.

[0071] The self-calibrating autofocus system can be implemented in a variety of configurations. Figures 5-7 are examples of alternative configurations. The configurations shown in Figures 5-7 depict the components of the autofocus system 200 of Figure 2A with any added components labeled with different numbers. Additionally, the controller 220 and components 221 for sample imaging are not shown.

[0072] 5 is an example of an autofocusing system 500 configured to perform self-calibrating autofocusing within a microscope system (not shown) illustrating an alternative location for positioning the off-center aperture 502. The off-center aperture 502 is located between the first imaging lens 208 and the beam splitter 210, as opposed to after the beam splitter 210. The optical path shown in FIG. 5 is only a portion of the total light that is passed through the off-center aperture 502.

[0073] FIG. 6 is another example of an autofocusing system 600 configured to perform self-calibrating autofocusing. The light source 206 and the first imaging lens 208 can be configured to generate a light beam that fills the entrance pupil of the objective lens 202. Using a light beam that fills the entrance pupil of the objective lens 202 can reduce alignment errors and maximize sensitivity. The system 600 of FIG. 6 includes an off-center aperture 602 that is inserted above the second imaging lens 604 to control the direction and sensitivity of the beam translation. In an exemplary implementation, the off-center aperture 602 is switchable (optically or mechanically in a manner known to those skilled in the art) to provide a way to improve light throughput and accuracy in the step of finding the best focus position.

[0074] 7 is another example of an autofocusing system 700 configured to perform self-calibrating autofocusing. The autofocusing system 700 of FIG. 7 includes an off-axis imaging lens 702 and a beam expanding lens 704 positioned above an off-center aperture 706.

[0075] The off-axis imaging lens 702 is positioned off-axis such that only a portion of the light beam passes through the off-axis imaging lens 702. The light that passes through the off-axis imaging lens 702 is directed to a beam expanding lens 704. The light then proceeds to an off-center aperture 706, which further accentuates the lateral position of the beam.

[0076] It should be apparent to one skilled in the art that any combination of hardware and / or software may be used to implement the auto-focusing system 200 described herein. It should be understood that one or more of the processes, sub-processes, and process steps described in connection with Figures 2-7 may be implemented by hardware, software, or a combination of hardware and software on one or more electronic or digital control devices. The software may reside in a software memory (not shown) within a suitable electronic processing component or system, such as, for example, one or more of the functional systems, controllers, devices, components, modules, or sub-modules depicted diagrammatically in Figures 2-7. The software memory may include an ordered enumeration of executable instructions for implementing logical functions (i.e., "logic" which may be implemented in digital form, such as digital circuitry or source code, or in analog form, such as analog sources, such as analog electrical, sound, or video signals). The instructions may be executed within a processing module or controller (e.g., microscope controller 220 of FIG. 2) including, for example, one or more microprocessors, general purpose processors, processors, digital signal processors (DSPs), field programmable gate arrays (FPGAs), or combinations of application specific integrated circuits (ASICs). Furthermore, the schematic diagram illustrates a logical division of functions with physical (hardware and / or software) implementations that are not limited by the architecture or physical layout of the functions. The exemplary systems described herein may be implemented in various configurations and operate as hardware / software components within a single hardware / software unit or within separate hardware / software units.

[0077] The executable instructions may be implemented as a computer program product having instructions stored therein that, when executed by a processing module of an electronic system, direct the electronic system to perform the instructions. The computer program product may optionally be embodied in any non-transitory computer-readable storage medium for use by or in association with an instruction execution system, apparatus, or device, such as an electronic computer-based system, a processor-containing system, or other system that may selectively fetch instructions from and execute the instructions from the instruction execution system, apparatus, or device. In the context of this document, a computer-readable storage medium is any non-transitory means that may store a program for use by or in association with an instruction execution system, apparatus, or device. The non-transitory computer-readable storage medium may optionally be, for example, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device. A non-exhaustive list of more specific examples of non-transitory computer readable media includes an electrical connection having one or more wires (electronic), a portable computer diskette (magnetic), random access, i.e., volatile memory (electronic), read-only memory (electronic), erasable programmable read-only memory such as, for example, flash memory (electronic), compact disc memory such as, for example, CD-ROM, CD-R, CD-RW (optical), and digital versatile disc memory, i.e., DVD (optical).

[0078] It should also be understood that signal reception and transmission as used herein means that two or more systems, devices, components, modules, or sub-modules can communicate with each other via signals traveling through some type of signal path. The signals may be communication, power, data, or energy signals that may communicate information, power, or energy from a first system, device, component, module, or sub-module to a second system, device, component, module, or sub-module along a signal path between the first and second system, device, component, module, or sub-module. The signal path may include physical, electrical, magnetic, electromagnetic, electrochemical, optical, wired, or wireless connections. The signal path may also include additional systems, devices, components, modules, or sub-modules between the first and second system, device, component, module, or sub-module.

[0079] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

[0080] In the context of describing the present invention (particularly in the context of the claims below), the use of the terms "a" and "an" and "the" and similar designations should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended only to more clearly illuminate the disclosure and does not impose limitations on the scope of the disclosure, unless otherwise claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0081] Numerous modifications to the present disclosure will be apparent to those of skill in the art in light of the foregoing description. It is understood that the illustrated embodiments are illustrative only and should not be taken as limiting the scope of the present disclosure.

Claims

1. 1. A method of automatic focusing for an objective lens in a microscope, the method comprising: disposing an off-center aperture in an optical path between the objective lens and an image capture device; generating a reference calibration gradient for the objective lens that indicates a linear relationship between lateral position and z-position along a linear path along which an image translates on the x-y plane by determining a position on an x-y plane for each of a plurality of images captured by the image capture device at a series of z-positions of the objective lens, wherein one image of the plurality of images has a particular attribute corresponding to a best focus position, the optical path formed during capture of the plurality of images being at least partially blocked by the off-center aperture, and the particular attribute corresponding to the best focus position is selected from the group consisting of: highest pattern correlation with an original pattern using pattern recognition techniques, smallest size of a patterned image, smallest beam spot size, highest illumination, highest intensity, and highest contrast; autofocusing the objective lens based on at least the reference calibration gradient and the best focus position; A method comprising:

2. 10. The method of claim 1, further comprising disposing a beam splitter in the optical path between the objective lens and the image capture device, the beam splitter configured to reflect a portion of the light toward the objective lens and a reference surface and to pass a portion of the light reflected from the reference surface toward the off-center aperture.

3. The method of claim 1 or claim 2, wherein the multiple images comprise a beam spot in each image formed by projecting light reflected from a reference surface onto an image capture device.

4. The method of claim 1 or claim 2, wherein the multiple images comprise a pattern in each image formed by projecting patterned light reflected from a reference surface onto an image capture device.

5. The method of claim 1 or claim 2, wherein determining the position in the xy plane of each of the plurality of images comprises determining a centre of mass location of each image along an axis of lateral translation.

6. The method of claim 5 , wherein generating the reference calibration gradient comprises determining a linear relationship between the center of mass locations of the images and the corresponding z positions.

7. Positioning a sample well within the optical path of the objective lens; identifying a distance to move the objective lens to the best focus position for the sample well; Further comprising: The identifying step comprises: Capturing a reference image; identifying a lateral position of the reference image along the linear path along which the reference image is translated in the xy plane; calculating a distance to the best focus position based on the lateral position of the reference image and the reference calibration gradient; The method according to claim 1 .

8. positioning the objective lens at the best focus position; determining an offset from the best focus position to a sample focus position; positioning said objective lens at said best focus position plus said offset; The method of claim 1 or claim 2, further comprising:

9. moving the objective lens to the best focus position; capturing an image of the sample at each of a plurality of z positions in a direction toward the sample; determining a focus score for each sample image at each corresponding z-position based on any one or combination of focus metrics including spot diameter, intensity, mean intensity, maximum signal measure, standard deviation, variance, and covariance; plotting the focus scores for each sample image for each z position; Identifying the highest peak in the final score; positioning the objective lens at the z position corresponding to the highest peak; The method of claim 1 or claim 2, further comprising:

10. 1. A system for auto-focusing an objective lens in a microscope system, the system comprising: A light source; an image capture device configured to capture images of light reflection from a reference surface while the objective lens is moved through a series of z-positions along a z-axis; and an off-center aperture disposed in an optical path from the objective lens to the image capture device, the off-center aperture configured to block at least a portion of the light from the light source; a controller configured to control the light source, the image capture device, and the objective lens; Equipped with The controller is configured to perform a self-calibrating auto-focusing process, and performing the self-calibrating auto-focusing process comprises: generating a reference calibration gradient for the objective lens that indicates a linear relationship between lateral position along a linear path along which an image translates on the xy plane and z-position by determining a position on the xy plane for each of a plurality of images taken at a series of z-positions of the objective lens, wherein one image of the plurality of images has a particular attribute corresponding to a best focus position, the particular attribute corresponding to the best focus position being selected from the group consisting of: highest pattern correlation with an original pattern using pattern recognition techniques, smallest size of a patterned image, smallest beam spot size, highest illumination, highest intensity, and highest contrast; autofocusing the objective lens based on at least the reference calibration gradient and the best focus position; By,System.

11. 11. The system of claim 10, further comprising a beam splitter disposed in the optical path from the objective lens to the image capture device, the beam splitter configured to reflect a portion of the light from the light source toward the objective lens and the reference surface and to pass a portion of the light reflected from the reference surface toward the off-center aperture.

12. The system of claim 10 or claim 11, wherein the light source is configured to project a beam spot onto the image capture device.

13. The system of claim 10 or claim 11, wherein the light source is configured to project a pattern onto the image capture device.

Images