Patent Description:
Microscopes have evolved from standalone arrangements of optics that allow a user to view an image from individual slides, to high-throughput imaging systems that generate images of samples deposited in multiple sample wells formed in trays or plates (also known as "microwells," "microtiter," and "microplate"). The optics in microscope systems may be controlled by electromechanical devices under computer program control. Images may be collected using image capturing devices such as for example, cameras, charge-coupled device or other image capture devices.

Modern microscope systems may be used for detecting and measuring optical signal characteristics. In some systems, the sample wells may contain material, such as biological material, for example, that has been treated with a marker or a reactive component that causes an emission of light. The microscope may be used to detect intensity levels and patterns of light as opposed to simply capturing an image. As such, the microscope may use signal detector devices as well as image-capturing devices to obtain measurements of light levels. The detection component of the microscope may thus include, for example, photomultiplier tubes, photodetectors, and photodetector arrays, in addition to image capturing 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 places in the optical path between the light source and the sample, or between the sample and the detection component to either excite the sample in a desired manner, or to measure selected optical characteristics including intensities at certain wavelengths and other characteristics.

In capturing or discerning patterns in images, or in measuring optical signals, optical devices, such as, lenses, mirrors, filters, etc., are disposed along an optical path from the light source to the image capturing device. An imaging lens is adjusted to focus the light source on an image plane of the image capturing device when the objective lens is in focus on the reference surface. A lack of focus appears in images as a blurring or fuzziness. Similarly, optical measurements taken from an unfocused objective may not yield accurate intensity levels and pixels that may be brighter or less bright than they are supposed to be due to the spreading of the image when de-focused. The objective lens should typically be positioned to focus on the sample before imaging or optical measurements are performed by a microscope system.

In high-throughput microscope systems, where a 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 many times as quickly as possible. The focusing process should also be sensitive and accurate while adding as little complexity as possible, and without perturbing the sample. The focusing process should also be an autofocusing process, or a process that is performed with as little involvement by the user as possible. Fluorescence microscopes are used in analyzing biological samples tagged with one or more fluorophores. It is important to avoid causing photo damage to the fluorophore and the biological sample.

Microscope systems typically perform a hardware-based autofocus for each sample being processed. In a high-throughput microscope system, 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 autofocus system, a sample well is illuminated through an objective lens that is to be used in the imaging or the optical measurements to be performed on the sample in the sample well. The objective lens is positioned at a series of positions along the optical axis along which the objective lens travels while an image and pixel intensity is checked at each position. The optical axis along which the objective lens travels may be referred to as a z-axis and the motion of the objective lens may be referred to as a scan along a z-axis.

<FIG> is a schematic diagram of a microscope <NUM> illustrating operation of a process for performing a hardware-based autofocus of the microscope <NUM> for imaging on a sample <NUM> in a sample well <NUM> disposed on a plate <NUM>. The microscope <NUM> in <FIG> includes main components for autofocus: an objective lens <NUM>, a detector <NUM>, a beam splitter <NUM>, and a light source <NUM>. The microscope <NUM> may be implemented in a system that includes components configured to control the state and the motion of the objective lens <NUM> and the plate <NUM>. Such components may also be configured to control the state of the light source <NUM> and operation of the detector <NUM>. For example, the movement of the objective lens <NUM> along a z-axis may be controlled using a linear actuator having an electric motor under control of a computer program. The objective lens <NUM> is moved to positions (i.e. z-positions) on the z-axis. The detector <NUM> may be implemented as a charge-coupled device or other image capturing device also controlled to capture an image and to transfer the digital content of the image to a computer by a computer program.

In an example hardware-based autofocusing process, a user may be provided with a user interface for entering configuration data to configure the microscope according to a type of objective lens <NUM>, type of plate <NUM>, and light source <NUM>. The system may then position the objective lens <NUM> at a starting location on the z-axis in a suitable proximity to the plate <NUM>. The starting position may depend on the type of plate <NUM> as different plates may have a plate bottom surface at different positions along the z-axis. The objective lens <NUM> may also be controlled to move along a working distance, or a scan range, through which a best focus position can be found. The process typically involves positioning the objective lens <NUM> at each z-position in the scan range and capturing an image at each z-position.

The light from the light source <NUM> follows a first optical path at <NUM> from the light source <NUM> to the beam splitter <NUM>. The beam splitter <NUM> reflects part of the light towards the objective lens <NUM>, which directs the light along optical path <NUM> towards the sample well <NUM>. The light reflects off the top and/or bottom surfaces of the plate <NUM> back towards the objective lens <NUM> and through the beam splitter <NUM> along optical path <NUM> to the detector <NUM>. If the position of the objective lens <NUM> is sufficiently close to a position at which the objective lens <NUM> focuses on a reflective surface of the plate <NUM>, the light appears at the detector <NUM> as a beam spot having a diameter and an intensity. As the objective lens <NUM> is moved along the z-axis, the system controls the detector <NUM> to capture an image at each z-axis position. Each image is analyzed to determine a focus score based on a focus metric that may include for example, average intensity, a maximum signal, standard deviation, variance, or covariance, size, morphology, position, and others, of the pixels in each spot. The smallest and brightest spot is typically deemed to have the "best focus. " The focus score may be plotted against the z-position at which each image is captured in a graph <NUM>. In the graph <NUM> (see <FIG>), the smallest and brightest spots might form a peak at <NUM> for its intensity and z-position values. The graph <NUM> may also indicate the brightest pixels as peaks <NUM> and <NUM>. The z-position at the highest focus score (at <NUM> in <FIG>) is identified as being the best focus position.

It is noted that two peaks may be formed in the graph indicating reflections off the two surfaces on the plate <NUM>. Once the peak or peaks are identified and the corresponding z-position identified for each peak, the process identifies the best focus position as the z-position corresponding to a selected one of the peaks. That is, the best focus position is the position at which the spot formed by the light reflected off either the top or bottom surface of the plate <NUM> has the best focus score.

The best focus position in the context of the autofocus procedure described with reference to <FIG> is the z-position of the objective lens <NUM> that produces the sharpest and clearest projection of a light beam reflected from a reference surface. In the system shown in <FIG>, the reference surface is either the top or bottom surface of the plate <NUM>. In order to image with good focus on the sample (<NUM> in <FIG>), such as for example, a collection of cells, the objective lens <NUM> should be moved to focus on the sample <NUM>. In some implementations, an offset along the z-axis may be defined according to a sample type, the objective lens, a plate type, and/or other suitable parameters. When a best focus position is determined, the objective lens <NUM> may be moved according to the offset to begin imaging. In some implementations, the system may perform an image-based autofocus after the hardware-based autofocus. An image-based focus involves moving the objective lens <NUM> along the z-axis while checking actual images of the sample until the sample is in focus.

The autofocus procedure described with reference to <FIG> may involve a hardware-based autofocus to find the reference surface on the plate <NUM> and either a move by an offset to focus on the sample, or an image-based focus to find the sample <NUM>. The hardware-based autofocus or the image-based autofocus, or both, may need to be performed each time a new sample well is presented for imaging even though the objective lens is not replaced. The microplates on which the sample wells are disposed may not be perfectly flat and the thickness of the sample well bottoms may not be consistent. Different samples or different light sources may also call for a re-focus.

Current hardware-based autofocus procedures are slowed by the need to collect data points from each z-position in the working distance of the objective lens <NUM>. In systems in which a microscope is used to image a large number of samples, even a short delay in the focusing of the objective of the microscope may be too long. Other processes that are used come with trade-offs and other limitations, such as complexity, reduced accuracy, sensitivity to the surface reflecting the light bundle imaged during focusing, and high cost. <CIT> discloses a method of focusing of an optical imaging apparatus. The method comprises causing illumination of an object using an illuminating beam to thereby cause generation of a scattered beam. An optical-system, interchangeably referred as an optical-imaging apparatus is disclosed. An objective-lens is configured to project an illuminating beam upon an object to thereby cause generation of a scattered beam. Such illuminating beam may be an off-axis beam or a diffused-beam. For such purposes, an off-axis aperture may be disposed against a source of the optical-beam to generate an off-axis beam as the illuminating beam. <CIT> suggests a system in which the sample face is irradiated with light emitted from a first light source through a half mirror and an objective lens. The sample face is irradiated with light emitted from a second light source through a lens. Light irradiating the sample face side of the first light shielding plate out of the reflected light from the sample face is detected by the first light quantity detecting element and light irradiating the sample face side of the second light shielding plate is detected by the second light quantity detecting element.

According to one aspect, a method is provided to perform a self-calibrating autofocusing procedure for an objective lens in a microscope system. In an example method, a reference calibration slope is generated by determining respective positions 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 corresponding to a best focus position. An autofocusing procedure may then be performed on the objective lens based at least in part on the reference calibration slope and the best focus position.

In another aspect, the reference calibration slope indicates a linear relationship between the series of obj ective lens positions and the lateral positions on an image plane as the reference images translate in the image plane.

In another aspect, the reference image may be a beam spot reflected from a reference surface and projected on to an image-capturing device. Alternatively, the reference image may be a patterned image.

In another aspect, a system for autofocus is provided. One example of an autofocus system includes a light source, a decentered aperture, and optical components to form optical paths towards an image capturing device. The autofocus system may be connected to a microscope system with an optical path that includes the objective lens of the microscope system. The autofocus system may also operate as a sub-system of the microscope system. The autofocus system also includes a controller or operate using a controller that operates in the microscope system. The controller may include storage for machine instructions that, when executed by a processor, performs a self-calibrating autofocus method.

The drawings illustrate the design and utility of embodiments of the disclosed inventions, in which similar elements are referred to by common reference numerals. These drawings are not necessarily drawn to scale. These drawings depict only typical embodiments of the disclosed inventions and are not therefore to be considered limiting of its scope.

All numeric values are herein assumed to be modified by the terms "about" or "approximately," whether or not explicitly indicated, wherein the terms "about" and "approximately" generally refer to a range of numbers that one of skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In some instances, the terms "about" and "approximately" may include numbers that are rounded to the nearest significant figure.

In describing the depicted embodiments of the disclosed inventions illustrated in the accompanying figures, specific terminology is employed for the sake of clarity and ease of description. However, the disclosure of this patent specification is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents that operate in a similar manner. It is to be further understood that the various elements and/or features of different illustrative embodiments may be combined with each other and/or substituted for each other wherever possible within the scope of this disclosure and the appended claims.

Various embodiments of the disclosed inventions are described hereinafter with reference to the figures. It should be noted that the figures are not drawn to scale and that elements of similar structures or functions are represented by like reference numerals throughout the figures. They are not intended as an exhaustive description of the invention or as a limitation on the scope of the disclosed inventions, which is defined only by the appended claims and their equivalents. In addition, an illustrated embodiment of the disclosed inventions needs not have all the aspects or advantages shown. For example, an aspect or an advantage described in conjunction with a particular embodiment of the disclosed inventions is not necessarily limited to that embodiment and can be practiced in any other embodiments even if not so illustrated.

Described below are systems and methods for determining a best focus position for an objective lens in a microscope system. The method(s) and system(s) described below disclose a calibration process that generates a reference slope corresponding to various known parameters. Specific to the present inventive concept(s), a decentered aperture is utilized to provide a sampling of the wavefront at a plurality of z positions that is strategically mapped to form a calibration reference slope. This reference slope derived through the calibration process may then be advantageously used to determine an offset from the best focus position, as will be discussed in further detail below. This approach allows the system to rapidly calibrate and return to the best focus during image acquisition, minimizing errors and improving throughput.

As used in the description below, the term "best focus position" shall mean the z-position of the objective lens at which the objective lens provides the best focus on a reference image projected on a reference surface. As such, the best focus position may not be the position at which the objective lens focuses on a 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 procedures or optical measurement procedures, an offset from 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, a hardware focus may be performed similar to that described above with reference to <FIG>. The advantage to starting such a hardware-based focus from close proximity to the best focus position is that fewer z-positions are used than with the procedure described above that does not start from a best focus position.

As used in the description below, the terms "imaging," "image capturing," "capture of images," or "detecting an image" shall refer to any process for collecting optical data from an image capturing device. The image may be a reference image for obtaining a reference calibration slope, an image of a sample, including capturing a digital image for storage, or measuring optical characteristics such as, intensity, color, or other types of data.

As used in the description below, the term "sample well" shall refer to any suitable structure for containing a sample to be presented for imaging or for obtaining an optical measurement. The sample well may include for example, a recessed structure formed on a microwell plate as defined below in which a sample may be deposited. The sample well may also include as the term is defined herein a slide with or without a cover or any other suitable structure for containing a sample.

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

As briefly discussed above, the present invention relates to systems and methods for calibrating a best focus position for an objective lens in a microscope system, and then to use the best focus position calibration to focus the objective lens more quickly and efficiently than with conventional autofocus techniques. When a sample is positioned for measurements using the calibrated objective lens and after the objective lens may have been re-positioned, the best focus position calibration is used to find the position of the objective lens relative to its best focus position. To focus on a sample, the objective lens may be moved to the best focus position or a predetermined offset from best focus.

The calibration of the best focus position for a given objective lens involves determining a relationship between the positions on the axis of travel of the objective lens and a translation of an image (or reference image) on an image plane of an image capturing device. An autofocus sub-system or module in a microscope system may include a suitable light source, a decentered aperture, a reference surface, imaging optics, and an image-capturing device. The reference surface may be a plate (i.e. microplate, microwell plate, microtiter, etc.) that may be used to hold sample wells that contain samples on which optical measurements will be performed. The components may be arranged to illuminate the reference surface with the light source through the objective lens of the microscope system. The optical components project a reference image on the image-capturing device as shown for example in <FIG> and <FIG>. The decentered aperture is positioned and sized to sample a portion of the total light through the entrance pupil of the objective to project on the image-capturing device as, for example, a beam spot. As the objective lens is moved along its axis of travel, the beam spot translates linearly and changes focus. This pattern is strategically utilized to create a reference slope that maps a position of the beam spot at a plurality of z positions, as will be discussed in further detail below.

<FIG> is an example implementation of an autofocus system <NUM> for calibrating a best focus position and autofocusing an objective lens <NUM> for a microscope system. The autofocus system <NUM> includes an image-capturing device <NUM>, a light source <NUM>, a first imaging lens <NUM>, a beam splitter <NUM>, a second imaging lens <NUM>, and an autofocusing aperture <NUM>. The objective lens <NUM> is a component of the microscope system configured to perform imaging and/or optical measurements on samples that may be deposited into a sample well <NUM>. Other components of the microscope system include any sample holding structure supporting the plate <NUM> with the reference surfaces 212a, 212b.

The autofocus system <NUM> in <FIG> is implemented as a module or a sub-system of the microscope system. Other components that the microscope system uses for imaging samples, such as for example, excitation light source, filters, beam splitters, and sample image capturing device, are represented in <FIG> as sample imaging components <NUM>. The sample imaging components <NUM> may include lenses, filters, or other optical devices, for example, that form optical paths that include the objective lens <NUM> and the plate <NUM> when the microscope system is used to image samples. The microscope system <NUM> may also use a different light source or a different sample image-capturing device based on the type of imaging or measurement being performed. The optical devices may be inserted below the objective lens in <FIG> at 221a above the beam splitter <NUM>, or at 221b above the decentered aperture <NUM>. The sample well <NUM> in the example shown in <FIG> is formed on a plate <NUM>, which provides a bottom reference surface 212a and a top reference surface 212b for the autofocus procedure.

Once a self-calibration of the best focus position is performed for a given objective lens <NUM>, the process of focusing that objective lens <NUM> in subsequent imaging or optical measurements may be performed with minimal further imaging. The calibration of the best focus position may be stored as a reference calibration slope, which may be stored or included with data characterizing the objective lens <NUM> in a system data storage system <NUM>.

The first imaging lens <NUM> collimates a light from the light source <NUM> along optical path <NUM> and passes the collimated light to the beam splitter <NUM>. The beam splitter <NUM> reflects a portion of the light towards the objective lens <NUM> along optical path <NUM>, and towards the plate <NUM> on optical path <NUM>. The plate <NUM> reflects the light back to the objective lens <NUM> and towards the beam splitter <NUM> on optical path <NUM>. The beam splitter <NUM> passes a portion of the light along optical path <NUM> towards the decentered aperture <NUM>. The light passes through an off-centered opening on the aperture <NUM> that is smaller than the total light beam impinging on the aperture <NUM>. The remaining portion of the aperture <NUM> occludes the part of the light beam that is not passed through the aperture. The light passing through the aperture <NUM> is directed to the second imaging lens <NUM> and to the image-capturing device <NUM>. The decentered aperture <NUM> operates by sampling a portion of the planar wavefront from the objective lens <NUM>. The sampled portion of light is focused by the second imaging lens <NUM> and directed towards the detector but constrained by the decentered aperture as an asymmetric marginal ray. This allows viewing the position of the light and best focus without changing any of the component setup. It should be appreciated that different decentered apertures having different sizes and/or positions may be used to allow for adjustment of sensitivity or for different sizes of the pupil diameter of the objective lens <NUM>.

It is noted that the optical paths <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> shown in <FIG> only show light along the optical paths that forms the light beam impinging on the image-capturing device <NUM>. The portion of the light not shown is the portion of the light that is occluded by the occluding portion of the autofocusing aperture <NUM>.

The objective lens <NUM> is configured to move along the optical paths <NUM> and <NUM> on a z-axis (shown in <FIG>), which is perpendicular to an x-y plane along which the plate <NUM> extends. The description below references positions of the objective lens as being on a z-axis, and positions of the reference images, or lateral positions, as being on an x-y plane as a way of providing clarity. The reference images translate along a linear path on the image plane. This linear path is referred to as the y-axis. It is to be understood that the use of the z-axis or the y-axis to provide a spatial reference is not intended to be limiting. Any suitable coordinate system may be used. It is further noted that example implementations may involve an objective lens <NUM> that travels in a non-vertical direction.

The plate <NUM> may include a sample well <NUM> as shown in <FIG>, which may be positioned for the imaging of a sample that may be deposited therein according to the normal functions and operation of the microscope system. In the example system shown in <FIG>, the plate <NUM> has a first surface 212a and a second surface 212b, which is the bottom surface of the sample well. The first surface 212a and/or the second surface 212b may be at least partially reflective and thereby provide a reflective reference surface to use during a self-calibrating autofocus procedure. The reference surface may also be provided on a cover slip, or on surfaces of a slide, or other planar material disposed in the optical path in proximity to the bottom surface of the sample well <NUM>.

The objective lens <NUM> may be moved along the z-axis using a linearly actuating motor controlled by the controller <NUM>. The objective lens <NUM> is represented schematically in <FIG> as including the linearly actuating motor that moves the objective lens <NUM>. The objective lens <NUM> includes selected optics configured to focus light from the light source on the sample <NUM> when the microscope system <NUM> is controlled to image on a sample. During the autofocus procedure, the objective lens <NUM> is controlled to focus on the reference surface (212a or 212b on the plate <NUM>). In some implementations, the motor that moves the objective lens <NUM> may be a stepper motor or a servomotor with a linear actuator.

The light along the optical path passing through the decentered aperture <NUM> travels through the second imaging lens <NUM> to the image capturing device <NUM> where a projected source is imaged on a detector plane. When defocused, the light beam on the image capturing device <NUM> is spread in size, has a lower intensity, and/or a low contrast. When the objective lens <NUM> is in focus, the reference image is captured at a maximum intensity, at a smallest size, and its highest contrast. The process of focusing the objective lens <NUM> involves moving the objective lens <NUM> to find the best focus position on the z-axis. Each beam spot in each reference image captured at each objective lens <NUM> z-position, appears on a position in the image plane that is offset from the spot position on the previous images.

In example implementations, the image capturing device <NUM> in the autofocusing system <NUM> may be a linear array detector, a charge-coupled device, a position sensitive diode, a <NUM>-D sensor array as the image capturing device, or any suitable device that may be controlled by a controller <NUM> to capture images of a reference image as the objective lens <NUM> is controlled to move to a series of z-positions. The light source <NUM> in the autofocusing system <NUM> may be any suitable light emitting device, such as a laser, a light emitting diode (LED) or an LED array, an incandescent light, a fluorescent light source, infrared light or the like.

The controller <NUM> may be implemented using any computer-programmable system having a hardware interface connected to at least the image capturing device <NUM> and the motor configured to move the objective lens <NUM>. In some implementations, the controller <NUM> may also be a component of the microscope system <NUM> for which the objective lens is being autofocused. The self-calibrating autofocusing procedure may be a function stored as software in the data storage medium <NUM> to which the controller <NUM> has access.

The self-calibration and autofocusing procedure for a given objective lens involves performing a first process of obtaining the reference calibration slope. The reference calibration slope may then be used to autofocus the objective lens thereafter. The flowchart in <FIG> illustrates operation of an example method <NUM> for autofocusing an objective lens by first obtaining a reference calibration slope at <NUM> and by using the reference calibration slope at <NUM> to determine the best focus position of the objective lens <NUM>. The objective lens <NUM> may then be moved to focus on a sample using an offset from the best focus position, or other methods, such as performing a hardware-based focus similar to the hardware-based method described above with reference to <FIG> on the sample.

Referring to <FIG> (for components referenced below) and 2B, a self-calibrating and autofocusing procedure may be initiated when an objective lens <NUM> is inserted for use in a microscope system <NUM> at step <NUM>. At step <NUM>, the system may perform initiation or setup functions for using the specific objective lens <NUM> that has been selected. One initiation step may include determining if a reference calibration slope has been generated for the objective lens <NUM> at decision block <NUM>. If no reference calibration slope has been generated (indicated by the 'NO' path to method <NUM>), a starting point location is determined for an autofocus procedure at step <NUM>.

When an objective lens <NUM> has been inserted, or installed, in a microscope system, the position of the objective lens <NUM> along the z-axis is typically not known to the system. The system <NUM> may perform a home function that positions the objective lens <NUM> in a known position based on a home structure that may be sensed by the objective lens <NUM> or a sensor affixed to the structure of the objective lens <NUM> as it moves. In other implementations, the objective lens <NUM> may be positioned at its furthest location from the plate <NUM>.

Once the objective lens <NUM> is in a known or home position, the controller <NUM> may position the objective lens <NUM>, at step <NUM>, a predetermined distance away from the home position in order to begin an autofocus procedure. The predetermined distance may be a starting position stored in a data description of the objective lens <NUM>. The starting position may be a parameter that is used for all or most objective lenses.

At step <NUM>, a reference image is captured at a current position of the objective lens <NUM>. The objective lens <NUM> is then positioned at a series of z-positions to scan the reference surface as the objective lens is moved. The scan involves capturing a reference image at each z-position. The z-position is checked to determine if the scan is complete as shown in decision block <NUM>. The example in <FIG> for the check used in decision block <NUM> determines if the scan is complete by checking if a reference image has been captured for each z-position in the scan. The scan may be performed with a set number of z-positions or until a limit of travel on the objective lens is reached. If the scan is not complete, the objective lens <NUM> is moved to the next z-position at step <NUM> (along the NO decision path). At step <NUM>, a reference image is captured at the next z-position. If the scan is complete, the reference images captured at each z-position are analyzed (the YES path of decision block <NUM>) at step <NUM>.

<FIG> is a schematic representation of an example of a plurality of images <NUM> illustrating a lateral translation (along a y-axis) of a beam spot 302a-o overlaid on a representation of a linear array detector <NUM> during a calibration of a best focus position for an objective lens in a microscope system.

The beam spots 302a-o in <FIG> are shown at <NUM> locations on the image plane of the linear array detector <NUM> for images captured at <NUM> corresponding z-positions of the objective lens <NUM> (<FIG>). It is noted that <FIG> shows <NUM> locations to illustrate the process, but at each time a single image plane is acquired per z-position. Although the illustrated embodiment shows <NUM> beam spots, it should be appreciated that any number of z positions and corresponding beam spots may be similarly used.

The beam spot <NUM> in each image is analyzed to determine the location of each beam spot. The determination of a centroid location for an imaged object is well known and need not be described in further detail. The y-axis location of the centroid of each beam spot <NUM> in each reference image is determined and associated with the z-position of the objective lens when each image was captured. <FIG> illustrates the lateral translation of the beam spot <NUM> as the reference images overlaid on one another. The beam spots <NUM> are located along the y-axis where the beam spots appear in the reference image in which each beam spot is captured.

Each beam spot 302a-o is shown in <FIG> as being unfocused at the initial y-axis locations, such as y = <NUM> through y = <NUM>. The level of focus is determined from measuring attributes of the beam spots <NUM>. The beam spots 302a-e for example have a lower intensity, which is indicated in the example in <FIG> by the lighter shade of each spot. At y = <NUM>, the beam spot 302f is more focused, which is determined by the smaller size and greater intensity of the beam spot image 302f. The greater intensity is indicated by the darkening shade of beam spot 302f. Beam spot <NUM> is the darkest (highest light intensity) and smallest spot in <FIG>.

As the objective lens <NUM> is moved further along the series of z-positions in the scan past y = <NUM>, the beam spots 302i-302o become larger and have a lighter shade, which indicate the reference images are defocusing. Referring to <FIG>, the best focus position may be determined, as shown at step <NUM>, by identifying the smallest and brightest beam spot, and setting the z-position at which that image was captured as the best focus position. In the example shown in <FIG>, the best focus position is the z-position corresponding to the y-position of beam spot <NUM>. As indicated above, other optical characteristics may be used to determine the best focus position besides intensity, and the example above should not be read as limiting. In addition, the determination of intensity for each beam spot <NUM> (in <FIG>) may be based on an average intensity of all pixels forming the spot, or other measures. In example implementations, the intensity or contrast may be used to determine a focus score for each spot.

At step <NUM>, further analysis compares the y-positions of the beam spots (302a-o in <FIG>) to the z-positions to determine a linear relationship between y and z positions. The linear relationship may then be stored as the reference calibration slope at step <NUM>.

While a plot of the data is not necessary to store the reference calibration slope, <FIG> illustrates an example of a plot of reference image position y vs. z objective position relative to the plate 212a. For convenience, the y and x axis have been shifted relative to the best focus position at the origin. As shown in <FIG>, the y-z points identified from the scan described with reference to <FIG> may lie on a curve C that may be approximated by a line L. The reference calibration slope for the objective lens <NUM> may be indicated as being the linear expression for line L (i.e. y = mz + B), which in the illustrated example is y = <NUM>. 37z + <NUM>. For the objective lens <NUM>, a reference image, such as a beam spot <NUM>, may be captured wherever the objective lens <NUM> is positioned. The position of the beam spot <NUM> on the y-axis may be determined. The current distance from the best focus position may then be determined using y = <NUM>. 37z + <NUM> and solving for z from one or more reference images within the linear range of the system.

The reference calibration slope preferably is a non-zero slope m. The reference calibration slope of m = <NUM> would result if no decentered aperture or imaging lenses are used in the autofocus system. A flat horizontal line would not allow for a determination of the best focus position because the beam spot would be in the same position in every reference image regardless of the position of the objective lens. The reference calibration slope may be adjusted by selecting a decentered aperture according to aperture size and location of the light passing portion of the aperture. Imaging lenses may also be added to the autofocus system before or after the decentered aperture to adjust the magnitude of the reference calibration slope. It should also be noted that alignment of the sensor to the trajectory of the reference image position on the sensor may be easily set by orientation of the decentered aperture relative to the sensor.

Referring back to <FIG> at decision block <NUM>, the objective lens inserted at step <NUM> may have already been calibrated for a best focus position. If so, a reference calibration slope is found for the objective lens <NUM> (the 'YES' decision path is followed) and method <NUM> is performed to obtain a focus. The reference calibration slope for the objective lens <NUM> is retrieved at step <NUM> and moved to a starting position at step <NUM>. At step <NUM>, an image is captured at the starting position. At step <NUM>, the image is analyzed to determine a position of the centroid of the beam spot in the image on the y-axis. The y-position of the image is then used in the reference calibration slope to determine the current distance between the current position of the objective lens <NUM> and the best focus position at step <NUM>.

The sample imaging components <NUM> (in <FIG>) of the microscope system may now be deployed to image a sample using the objective lens <NUM>. The objective lens <NUM> may be moved to focus on the sample at step <NUM> by moving to the best focus position and a predetermined offset along the z-axis. 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 an objective, sample, or longitudinal color offset. The objective lens may also be controlled to move to a predetermined offset by shifting the target reference image away from best focus and scaling the shift by the calibration slope.

Some optical measurements may require higher sensitivity to defocus. As noted above, the objective lens may be focused on a sample using a hardware-based focus as described above with reference to <FIG> but by starting from the best focus position determined using method <NUM> in <FIG>. Combining the profile method described with reference to <FIG> with the predictive nature of the decentered aperture and calibration slope would improve the speed of the method by defining a smaller search range. The smaller search range may be based on one or more measurements predicting the appropriate objective positions while maximizing sensitivity to defocus.

Referring back to <FIG>, if a more precise distance to the best focus position is needed, the objective lens may be moved to several different z-positions and steps <NUM>-<NUM> may be repeated for each z-position to which the objective lens <NUM> is moved. In this way, the linear relationship defining the reference calibration slope may be confirmed as well as finding best focus based the focus metric around best focus by fitting, interpolating, or other methods to improve resolution to defocus. This process is illustrated with reference to <FIG> show graphical representations of images <NUM>, <NUM>, <NUM> of beam spots <NUM> captured with the objective lens at different z-positions using an example of method <NUM> in <FIG>. Each spot <NUM> in <FIG> is shown with a centroid <NUM> as a smaller spot generally in a center of the spots. The image <NUM> in <FIG> shows the beam spot <NUM> at or near the best focus position. The image <NUM> in <FIG> shows the beam spot <NUM> in a y-position indicating the objective lens <NUM> is defocused in one direction away from best focus. The image <NUM> in <FIG> shows the beam spot <NUM> in a y-position indicating the objective lens <NUM> is defocused in the opposite direction of the beam spot <NUM> in <FIG> away from best focus. Images of three beam spots are shown in <FIG> 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, and clips the full dynamic range of the the measurable defocus of the objective lens. In example implementations, many more images of beam spots may be captured and analyzed to improve resolution, particularly for smaller ROIs.

The beam spots <NUM> in <FIG> may be analyzed to determine a best focus position and a calibration slope based on the scan of the three beam spots. The best focus position may be found by interpolating, fitting, or picking the peak focus metric from the three beam spot scans and then compared with the original best focus position. The calibration slope found by analyzing the three beam spot scan may also be compared with the original reference calibration slope. The best focus positions and the reference calibration slopes 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.

It is noted that the reference image may be a beam spot or a patterned image. The light source <NUM> may be configured to generate a white light, or any other light of a suitable wavelength as a simple light beam or as a patterned image. <FIG> is an example of a patterned image that may be projected on the reference surface 212a or 212b and detected by the image capturing device as a reference image. If a patterned image is used, the best focus may be determined by analyzing the reference images using preferred data transformations, such as, for example, a Fourier transformation and then calculating the correlation. The patterned image may then be analyzed for optical attributes correspond to a best focus similar to the techniques described above for beam spots. The lateral position may be determined as either a centroid or a brightest pixel, and a highest contrast may be a preferred image attribute to determine best focus. The patterned image may also be analyzed using pattern recognition methods where a best pattern match to the original pattern indicates a best focus.

A self-calibrating autofocusing system may be implemented in a variety of configurations. <FIG> are examples of alternative configurations. The configurations shown in <FIG> depict the components of the autofocus system <NUM> in <FIG> with any added components labeled with different numbers. In addition, the controller <NUM> and the components for sample imaging <NUM> are also not shown.

<FIG> is an example of an autofocus system <NUM> , not falling under the scope of the claims, which is configured to perform self-calibrating autofocusing in a microscope system (not shown) illustrating another location in which to position a decentered aperture <NUM>. The decentered aperture <NUM> is disposed between the first imaging lens <NUM> and the beam splitter <NUM> as opposed to after the beam splitter <NUM>. The optical paths shown in <FIG> are only the portion of total light passed through the decentered aperture <NUM>.

<FIG> is another example of an autofocusing system <NUM> configured to perform self-calibrating autofocusing. The light source <NUM> and first imaging lens <NUM> may be configured to generate a light beam that fills an entrance pupil of the objective lens <NUM>. Using a light beam that fills the entrance pupil of the objective lens <NUM> may reduce alignment errors and maximize sensitivity. The system <NUM> in <FIG> includes a decentered aperture <NUM> inserted above a second imaging lens <NUM> to control the direction and sensitivity of the beam translation. In an example implementation, the decentered aperture <NUM> is switchable (optically or mechanically in ways known to those of ordinary skill in the art) to provide a way of increasing light throughput and precision in finding the best focus position.

<FIG> is another example of an autofocus system <NUM> configured to perform self-calibrating autofocusing. The autofocus system <NUM> in <FIG> includes an off-axis imaging lens <NUM>, and a beam magnifying lens <NUM> positioned above a decentered aperture <NUM>.

The off-axis imaging lens <NUM> is positioned off-axis so that only a portion of the light beam passes the off-axis imaging lens <NUM>. The light passing the off-axis imaging lens <NUM> is directed to the beam magnifying lens <NUM>. The light then passes to the decentered aperture <NUM>, which further emphasizes the lateral position of the beam.

It should be apparent to those who have skill in the art that any combination of hardware and/or software may be used to implement the autofocus system <NUM> described herein. It will be understood and appreciated that one or more of the processes, sub-processes, and process steps described in connection with <FIG> may be performed by hardware, software, or a combination of hardware and software on one or more electronic or digitally-controlled devices. The software may reside in a software memory (not shown) in 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 schematically depicted in <FIG>. The software memory may include an ordered listing of executable instructions for implementing logical functions (that is, "logic" that may be implemented in digital form such as digital circuitry or source code, or in analog form such as analog source such as an analog electrical, sound, or video signal). The instructions may be executed within a processing module or controller (e.g., the microscope controller <NUM> in <FIG>), which includes, for example, one or more microprocessors, general purpose processors, combinations of processors, digital signal processors (DSPs), field programmable gate arrays (FPGAs), or application-specific integrated circuits (ASICs). Further, the schematic diagrams describe a logical division of functions having physical (hardware and/or software) implementations that are not limited by architecture or the physical layout of the functions. The example systems described in this application may be implemented in a variety of configurations and operate as hardware/software components in a single hardware/software unit, or in separate hardware/software units.

The executable instructions may be implemented as a computer program product having instructions stored therein which, when executed by a processing module of an electronic system, direct the electronic system to carry out the instructions. The computer program product may be selectively embodied in any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as an electronic computer-based system, processor-containing system, or other system that may selectively fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions. In the context of this document, computer-readable storage medium is any non-transitory means that may store the program for use by or in connection with the instruction execution system, apparatus, or device. The non-transitory computer-readable storage medium may selectively 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 include: an electrical connection having one or more wires (electronic); a portable computer diskette (magnetic); a random access, i.e., volatile, memory (electronic); a read-only memory (electronic); an erasable programmable read only memory such as, for example, Flash memory (electronic); a compact disc memory such as, for example, CD-ROM, CD-R, CD-RW (optical); and digital versatile disc memory, i.e., DVD (optical).

It will also be understood that receiving and transmitting of signals as used in this document means that two or more systems, devices, components, modules, or sub-modules are capable of communicating with each other via signals that travel over some type of signal path. The signals may be communication, power, data, or energy signals, which 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 paths may include physical, electrical, magnetic, electromagnetic, electrochemical, optical, wired, or wireless connections. The signal paths may also include additional systems, devices, components, modules, or sub-modules between the first and second system, device, component, module, or sub-module.

The use of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

Claim 1:
A method of autofocus for an objective lens (<NUM>) in a microscope (<NUM>, <NUM>, <NUM>, <NUM>), comprising:
generating a reference calibration slope for the objective lens (<NUM>) 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, wherein an optical path formed during capture of the plurality of images is at least partially occluded by a decentered aperture (<NUM>, <NUM>, <NUM>); and
autofocusing the objective lens (<NUM>) based at least in part on the reference calibration slope and the best focus position,
characterized in that
a portion of the light towards the objective lens (<NUM>) and towards a reference surface (212a, 212b) is reflected by a beam splitter (<NUM>), wherein a portion of the light reflected from the reference surface (212a, 212b) is passed by the beam splitter (<NUM>) along an optical path (<NUM>) towards the decentered aperture (<NUM>, <NUM>, <NUM>).