Patent Publication Number: US-11656429-B2

Title: Systems, devices, and methods for automatic microscopic focus

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This is a continuation of U.S. application Ser. No. 16/715,571, filed Dec. 16, 2019, which is a continuation of U.S. application Ser. No. 16/275,177, filed Feb. 13, 2019, now U.S. Pat. No. 10,509,199, issued Dec. 17, 2019, which is a continuation of U.S. application Ser. No. 15/920,850, filed Mar. 14, 2018, now U.S. Pat. No. 10,247,910, issued Apr. 2, 2019, which are incorporated by reference in their entireties. 
    
    
     TECHNICAL FIELD 
     The present disclosure relates to image-based mechanisms for automatic microscopic focus. 
     BACKGROUND 
     Most specimens that are observed with a microscope have small variations in height across their surfaces. While these variations are frequently not visible to the human eye, they can cause images of a portion of a specimen captured by a microscope to be out of focus. 
     The range in which a microscope can create a usable focused image is known as the depth of field. The microscope must keep a portion of a specimen within its depth of field to generate useful images. However, when transitioning from observing a first portion of a specimen to observing a second portion of the specimen, the small variations in height of the specimen may cause the second portion to be outside the depth of field. 
     Different sharpness measurements such as image contrast, resolution, entropy and/or spatial frequency content, among others, can be used to measure the quality of focus of images captured by a microscope. Generally, when a specimen is in focus, the captured image will exhibit the best sharpness quality (e.g., large contrast, a high range of intensity values and sharp edges). The different sharpness measurements that can be used to determine when a specimen is in focus usually require capturing a series of images and increasing or decreasing the distance between the microscope objective lens and the specimen until the image appears in focus. This increases the total microscopic scan time of each specimen, making methods using such measurement prohibitively slow for high throughput scanning applications. 
     Accordingly, it is desirable to find a suitable in-focus plane of a specimen using a smaller number of images. 
     SUMMARY 
     Systems, methods, and media for automatic microscopic focus are provided. 
     In some embodiments, systems for automatically focusing a microscope are provided, the systems comprising: an objective; a stage for positioning a specimen on a first image forming conjugate plane; a first camera, configured for focusing, positioned on a second image forming conjugate plane; a second camera, configured for focusing, positioned at an offset distance from the second image forming conjugate plane; a primary illumination source that emits light in a first wavelength range, wherein the emitted light is received by the first camera; a secondary illumination source that emits light in a second wavelength range which is different from the first wavelength range and that projects light through a focusing pattern that is positioned on a third image forming conjugate plane, wherein the projected light is received by the second camera; and a hardware processor coupled to the first camera and the second camera that is configured to: determine, using the first camera, when a specimen is in focus based on a sharpness value; determine, using the second camera, a sharpness setpoint for the specimen when the specimen is determined to be in focus for the first camera; after movement of the specimen, determine, using the second camera, a first sharpness value of the specimen; determine whether the first sharpness value of the specimen is higher or lower than the sharpness setpoint; and adjust a distance between the objective and the stage so that a second sharpness value of the specimen determined using the second camera corresponds to the sharpness setpoint. 
     In some embodiments, systems for automatically focusing a microscope are provided, the systems comprising: an objective; a stage for positioning a specimen on a first image forming conjugate plane; a first camera, configured for taking images of the specimen when the specimen is determined to be in focus, positioned on a second image forming conjugate plane; a second camera, configured for focusing, positioned on a third image forming conjugate plane; a third camera, configured for focusing, positioned at an offset distance from the third image forming conjugate plane; a primary illumination source that emits light in a first wavelength range, wherein the emitted light is received by the first camera; a secondary illumination source that emits light in a second wavelength range which is different from the first wavelength range and that projects light through a focusing pattern that is positioned on a fourth image forming conjugate plane, wherein the projected light is received by the second and third camera; and a hardware processor coupled to the second camera and the third camera that is configured to: determine, using the second camera, when a specimen is in focus based on a sharpness value; determine, using the third camera, a sharpness setpoint for the specimen when the specimen is determined to be in focus for the second camera; after movement of the specimen, determine, using the third camera, a first sharpness value of the specimen; determine whether the first sharpness value of the specimen is higher or lower than the sharpness setpoint; and adjust a distance between the objective and the stage so that a second sharpness value of the specimen determined using the third camera corresponds to the sharpness setpoint. 
     In some embodiments, methods for automatically focusing a microscope are provided, the methods comprising: positioning a specimen on a stage on a first image forming conjugate plane; positioning a first camera, configured for focusing, on a second image forming conjugate plane; positioning a second camera, configured for focusing, at an offset distance from the second image forming conjugate plane; emitting, from a primary illumination source, light in a first wavelength range, wherein the emitted light is received by the first camera; emitting, from a secondary illumination source, light in a second wavelength range which is different from the first wavelength range, wherein the light is projected through a focusing pattern that is positioned on a third image forming conjugate plane, wherein the projected light is received by the second camera; determining, by a hardware processor using the first camera, when a specimen is in focus based on a sharpness value; determining, by the hardware processor using the second camera, a sharpness setpoint for the specimen when the specimen is determined to be in focus for the first camera; after movement of the specimen, determining, by the hardware processor using the second camera, a first sharpness value of the specimen; determining, by the hardware processor, whether the first sharpness value of the specimen is higher or lower than the sharpness setpoint; and adjusting, by the hardware processor, a distance between an objective and the stage so that a second sharpness value of the specimen determined using the second camera corresponds to the sharpness setpoint. 
     In some embodiments, methods for automatically focusing a microscope are provided, the methods comprising: positioning a specimen on a stage on a first image forming conjugate plane; positioning a first camera, configured for taking images of the specimen when the specimen is determined to be in focus, on a second image forming conjugate plane; positioning a second camera, configured for focusing, on a third image forming conjugate plane; positioning a third camera, configured for focusing, at an offset distance from the third image forming conjugate plane; emitting, from a primary illumination source, light in a first wavelength range, wherein the emitted light is received by the first camera; emitting, from a secondary illumination source, light in a second wavelength range which is different from the first wavelength range, wherein the light is projected through a focusing pattern that is positioned on a fourth image forming conjugate plane, wherein the projected light is received by the second and third camera; determining, by a hardware processor using the second camera, when a specimen is in focus based on a sharpness value; determining, by the hardware processor using the third camera, a sharpness setpoint for the specimen when the specimen is determined to be in focus for the second camera; after movement of the specimen, determining, by the hardware processor using the third camera, a first sharpness value of the specimen; determining, by the hardware processor, whether the first sharpness value of the specimen is higher or lower than the sharpness setpoint; and adjusting, by the hardware processor, a distance between the objective and the stage so that a second sharpness value of the specimen determined using the third camera corresponds to the sharpness setpoint. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    shows an example of an automatic focus system in accordance with some embodiments of the disclosed subject matter. 
         FIG.  2    shows an example of an illumination unit in accordance with some embodiments of the disclosed subject matter. 
         FIG.  3    shows an example of a focusing unit in accordance with some embodiments of the disclosed subject matter. 
         FIG.  4    shows an example of an optical pathway in accordance with some embodiments of the disclosed subject matter. 
         FIG.  5    shows an example of a sharpness curve for a primary focusing camera in accordance with some embodiments of the disclosed subject matter. 
         FIG.  6    shows an example of a sharpness curve for an offset focusing camera in accordance with some embodiments of the disclosed subject matter. 
         FIG.  7    shows an example of a sharpness curves for an offset focusing camera at different distances from an image-forming conjugate plane in accordance with some embodiments of the disclosed subject matter. 
         FIG.  8    shows an example of a flow chart of a process for performing automatic focus using an automatic focus system, such as the system illustrated in  FIG.  1   , in accordance with some embodiments of the disclosed subject matter. 
     
    
    
     DETAILED DESCRIPTION 
     In accordance with some embodiments of the disclosed subject matter, mechanisms (which can include systems, methods, devices, apparatuses, etc.) for automatic microscopic focus of specimens are provided. 
       FIG.  1    illustrates an example  100  of an automatic focus system according to some embodiments of the disclosed subject matter. At a high level, the basic components of automatic focus system  100 , according to some embodiments, include an illumination unit  200  for providing light, a focusing unit  300  for finding the in-focus plane of a specimen, a vertical illuminator  13 , an imaging camera  5 , an objective  25 , a stage  30 , and a control system  108  comprising hardware, software, and/or firmware. 
     Automatic focus system  100  can be implemented as part of any suitable type of microscope. For example, in some embodiments, system  100  can be implemented as part of an optical microscope that uses transmitted light or reflected light. More particularly, system  100  can be implemented as part of the nSpec® optical microscope available from Nanotronics Imaging, Inc. of Cuyahoga Falls, Ohio Although the following description refers to a reflected light vertical illuminator  13 , the mechanisms described herein can be a part of microscopes that do not use a reflected light vertical illuminator. 
     According to some embodiments, the microscope can include, one or more objectives  25 . The objectives can have different magnification powers and/or be configured to operate with brightfield/darkfield microscopy, differential interference contrast (DIC) microscopy and/or any other suitable form of microscopy. The objective and/or microscopy technique used to inspect a specimen can be controlled by software, hardware, and/or firmware in some embodiments. 
     In some embodiments, a fine focus actuator  23  can be used to drive objective  25  in a Z direction towards and away from stage  30 . Fine focus actuator  23  can be designed for high precision and fine focus adjustment of objective  25 . Fine focus actuator  23  can be a stepper motor, servo motor, linear actuator, piezo motor, and/or any other suitable mechanism. For example, in some embodiments, a piezo motor can be used and can drive the objective 0 to 50 micrometers (μm), 0 to 100 μm, or 0 to 200 μm, and/or any other suitable range(s) of distances. 
     In some embodiments, an XY translation stage can be used for stage  30 . The XY translation stage can be driven by stepper motor, servo motor, linear motor, and/or any other suitable mechanism. 
     In some embodiments, focus unit  32 , comprising actuator  35 , can be used to adjust stage  30  in a Z direction towards and away from objective  25 . Actuator  35  can be used to make coarse focus adjustments of, for example, 0 to 5 mm, 0 to 10 mm, 0 to 30 mm, and/or any other suitable range(s) of distances. Actuator  35  can also be used to move stage  30  up and down to allow specimens of different thicknesses to be placed on the stage. Actuator  35  can also be used in some embodiments to provide fine focus of, for example, 0 to 50 μm, 0 to 100 μm, 0 to 200 μm, and/or any other suitable range(s) of distances. In some embodiments, focusing unit  32  can also include a location device  33 . The location device can be configured to store an absolute position of stage  30  (e.g., the position of the stage when a specimen is in focus), even upon reset and/or power cycling of autofocus system  100 . In some embodiments, the location device can be a linear encoder, a rotary encoder or any other suitable mechanism to track the absolute position of stage  30  with respect to the objective. 
     In some embodiments, automatic focus system  100 , when it is properly focused and aligned, can include a set of conjugate focal planes, for example an image-forming conjugate set, that occur along the optical pathway through the microscope. Each plane within the image-forming conjugate set is conjugate with the others in that set because the planes are simultaneously in focus and can be viewed superimposed upon one another when observing specimens through the microscope. The set of image-forming conjugate planes in automatic focus system  100  can include a primary focusing camera  72  image plane, an imaging camera  5  image plane, a focusing pattern  55  image plane, a field diaphragm (F-stop)  14  image plane and a specimen image plane. More specifically, all references herein to positioning a primary focusing camera  72  and imaging camera  5  on the image-forming conjugate planes refer to positioning the sensors within cameras  5  and  7  on the image-forming conjugate planes. 
     In some embodiments, focusing pattern  55  can be formed from opaque material, with a pattern cut out of the material. The cutout section of the material allows light to pass through to the specimen image plane, while the opaque material section blocks light from passing through. In other embodiments, focusing pattern  55  can be formed from clear material such as clear glass or clear plastic that has an opaque pattern thereon which causes an image to be projected on the specimen image plane by light passing through the clear glass or plastic. 
     In some embodiments, imaging camera  5  can include an image sensor  6  that is positioned on an image-forming conjugate plane of automatic focus system  100 . Imaging camera  5  can be used to capture images of a specimen once control system  108  determines that the specimen is in focus. Image sensor  6  can be, for example, a CCD, CMOS and/or any other suitable electronic device that allows images of a specimen to be captured and stored. 
     In some embodiments, control system  108 , comprising controller  110  and controller interface  107 , can control any settings of the components of automatic focus system  100  (e.g., actuators  35  and  23 , primary illumination source  65 , secondary illumination source  40 , focusing cameras  70  and  72 , stage  30 , focusing pattern  55 , imaging camera  5  and objective  25 ), as well as communications, operations (e.g., taking images, turning on and off an illumination source, moving stage  30  and objective  25 , storing different values associated with a specimen) and calculations (e.g., sharpness calculations) performed by, and between, components of the automatic focus system. Control system  108  can include any suitable hardware (which can execute software in some embodiments), such as, for example, computers, microprocessors, microcontrollers, application specific integrated circuits (ASICs), and digital signal processors (any of which can be referred to as a hardware processor), encoders, circuitry to read encoders, memory devices (including one or more EPROMS, one or more EEPROMs, dynamic random access memory (“DRAM”), static random access memory (“SRAM”), and/or flash memory), and/or any other suitable hardware elements. In some embodiments, individual components within automatic focus system  100  can include their own software, firmware, and/or hardware to control the individual components and communicate with other components in automatic focus system  100 . 
     In some embodiments, communication  120  between the control system (e.g., controller  110  and controller interface  107 ) and the components of automatic focus system  100  can use analog technologies (e.g., relay logic), digital technologies (e.g., using RS232, ethernet, or wireless) and/or any other suitable communication technologies. 
     In some embodiments, operator inputs can be communicated to the control system using any suitable input device (e.g., a keyboard, mouse or joystick). 
       FIG.  2    shows the general configuration of an embodiment of an illumination unit of the automatic focus system, in accordance with some embodiments of the disclosed subject matter. The illumination unit  200  can include two illumination sources, for example a primary illumination source  65  and a secondary illumination source  40 . The illumination sources can provide light beams in ranges of wavelengths that are different from each other. 
     In some embodiments, for example, primary illumination source  65  provides a light beam having a wavelength in the range of 451 to 750 nanometers (nm), while the secondary illumination source  40  provides a light beam having a wavelength that is higher or lower than the range of wavelengths used for the primary source. For example, the wavelength range of the primary illumination source  65  can be in the range of 550 to 750 nm and the wavelength range of the secondary illumination source can be in the range of 400 to 450 nm. Light of any wavelength range can be used for primary illumination source  65  as long as the value of the range is known and can be separated from other wavelengths using known filtering techniques. Similarly, light of any wavelength range can be used for secondary illumination source  40 , as long as the light is not in the same wavelength range as primary illumination source  65 . 
     In some embodiments, as shown in  FIG.  1   , primary illumination source  65  is positioned so that its light is transmitted in a horizontal direction towards vertical illuminator  13 . Primary illumination source  65  can include a focusing lens  49  (e.g., a double convex lens) for focusing the primary light beam. The secondary illumination source  40  can be positioned at a suitable distance below focusing pattern  55  located on image-forming conjugate plane  54 . The diameter of focusing pattern  55  (e.g., 5 mm) can be adjusted so that a projection of focusing pattern  55  is smaller than the field of view (FOV) of focusing cameras  70  and  72 . Focusing pattern  55  can be any suitable geometric shape for example, a circle, rectangle, triangle, or hexagon. Focusing pattern  55  can also include a series of discrete openings, so that when light is transmitted through the discrete openings, the lines and spaces are projected across the field of view. In some embodiments, the location of primary illumination source  65  and secondary illumination source  40  can be switched. 
     In some embodiments, automatic focus system  100  can be configured so that light from secondary illumination source  40  is continuously transmitted through focusing pattern  55  in order to continuously project the focusing pattern image on a specimen that can be captured by focusing cameras  70  and  72 . The continuous projection of the focusing pattern image can facilitate sharpness focus of a specimen, especially for transparent specimens or for specimens that lack any visually recognizable features. Focusing pattern  55  can be used instead of, or in addition to, a field diaphragm, for sharpness focusing. For example, automatic focus system  100 , in addition to focusing pattern  55 , can also include a field diaphragm (F-stop)  14  that can be located in the vertical illuminator  13 . Field diaphragm  14  can also be positioned on an image-forming conjugate plane of automatic focus system  100 . In some embodiments, field diaphragm  14  controls the diameter of light emitted by illumination source  65  and  40  and transmitted to objective  25 . More specifically, in some embodiments, by reducing the size of the field diaphragm, the diameter of the light passing through is reduced. This creates a dark outline around the image of the specimen received by focusing cameras  70  and  72  and can be used to adjust the focus of the specimen (e.g., by moving the specimen and objective closer together or farther apart). At the point of greatest measured sharpness, the specimen is considered to be in-focus and the field diaphragm can be opened to a larger size to allow imaging of the specimen by imaging camera  5 . Reducing the field diaphragm and returning it to its original size, however, takes time (e.g., 2-5 seconds) and can slow down the scanning process and throughput. 
     Focusing pattern  55  can be positioned on an any suitable image-forming conjugate plane of automatic focus system  100  (e.g., above secondary illumination source  40  (as shown in  FIG.  1   ), or at field diaphragm  14 ), as long as an appropriate filter is used, when necessary, to make sure that focusing pattern  55  is not projected onto imaging camera  5 . For example, if focusing pattern  55  is positioned on the field diaphragm  14  image forming conjugate plane, in place of field diaphragm  14 , then a filter would be necessary. In some embodiments, a band filter can be located on the field diaphragm image forming conjugate plane (in place of field diaphragm  14 ) and a focusing pattern in the form of a pattern cutout can be created in the band filter. More specifically, a band filter can be selected that transmits light in the same wavelength range of primary illumination source  65  (e.g., greater than 450 nm) and blocks light in the same wavelength range of secondary illumination source  40  (e.g., less than or equal to 450 nm), except in the focusing pattern  55  region. In other words, light in the same wavelength range of secondary illumination  40  source would be blocked except in the region of focusing pattern  55 , which would allow the light from secondary illumination  40  to be transmitted through to focusing cameras  70  and  72 . As described below, in connection with  FIG.  4   , optical filter  11  ensures that only light from the primary illumination source  65  is transmitted to imaging camera  5 . 
     Note that, in some embodiments, any suitable illumination source can be used with illumination unit  200 , such as a 400 nm ultraviolet collimated light-emitting diode (LED) for secondary illumination source  40  and a  5500 K white light collimated LED for primary illumination source  65 . 
     In some embodiments focusing lens  45  (e.g., a 60 mm focal length bioconvex lens) can be placed at a suitable distance between the secondary illumination source  40  and focusing pattern  55 . Further, another focusing lens  47  can be placed at a suitable distance on the other side of focusing pattern  55 . In some embodiments, the distance of the lenses  45  and  47  from focusing pattern  55  can be based on the optical characteristics of the microscope to ensure the focusing of the light and positioning of focusing pattern  55  to be in a conjugate image-forming plane. 
     In some embodiments, a dichroic  60  is placed in the optical pathway of both primary illumination source  65  and secondary illumination source  40  before the light travels to vertical illuminator  13 . Dichroic, as used herein, can refer to mirrors, beam splitters, filters or beam combiners that transmits light of a known, specified wavelength and combines the with a light of another known, specified wavelength. Note that a combination of the aforementioned devices can be used to reflect and transmit the desired illumination sources and wavelengths. In some embodiments, a dichroic having a specific cut-off wavelength is selected in order to reflect the wavelengths of light emitted by secondary illumination source  40  and to allow the wavelengths of light emitted from primary illumination source  65  to pass through. For example, if secondary illumination source  40  emits light in a wavelength range of 400-450 nm and primary illumination source  65  emits light in a wavelength range of 550-750 nm, then a 450 nm cutoff dichroic (i.e., a dichroic that reflects light with a wavelength of 450 nm and below and allows light with a wavelength greater than 450 nm to pass through thereby combining the beams) can be used to reflect light from secondary illumination source  40  and to allow light from primary illumination source  65  to pass through. Dichroic  60  can be designed for a 45° angle of incidence, so that rejected light from secondary illumination source  40  is reflected at an angle of 90° and travels parallel to the light path from primary illumination source  65 . 
     In some embodiments, primary illumination source  65  can be the light source used for imaging a specimen on imaging sensor  6  in imaging camera  5  and secondary illumination source  40  can be the light source used for imaging a specimen on focusing sensors  71  and  73  of focusing cameras  70  and  72 . 
     Note that, in some embodiments any suitable dichroic, illuminator, illumination source, focusing lens, sensor and focusing pattern can be used with illuminating unit  200 . In some embodiments, any suitable arrangement of these components can be used with illuminating unit  200 . In some embodiments, the components of illuminating unit  200  can be mounted to illuminator  13  in any suitable manner, such as by using guide rods in a similar manner to how focusing camera  72  is shown as being mounted to focusing housing  18  in  FIG.  3    (described below), in order to allow variable geometry. 
       FIG.  3    shows an example of a general configuration of an embodiment of a focusing unit of the automatic focus system, in accordance with some embodiments of the disclosed subject matter. The focusing unit  300  can include two cameras: a primary focusing camera  70  and an offset focusing camera  72 . These cameras can include, for example, a charged coupled device (CCD) image sensor, a CMOS image sensor and/or any other suitable image sensor that allows images of a specimen to be captured. In some embodiments, the captured image is stored and analyzed by control system  108 . 
     The focusing unit  300  can be mounted in an area between vertical illuminator  13  and imaging camera lens tube  10 . This area is known as infinity space. In some embodiments, the focusing unit  300  can be mounted in other locations using appropriate components to adapt the selected location to the optical characteristics of the system. 
     Primary focusing camera  70  can include a sensor  71  that is positioned on an image-forming conjugate plane of automatic focus system  100  (as represented for example by line  80 ). 
     An offset focusing camera  72  can include a sensor  73  that can be positioned at an offset to image-forming conjugate plane  80 . The offset can be either be in the positive direction  81  or the negative direction  79 . Offset focusing camera  72  can be located above or below primary focusing camera  70 . Offset focusing camera  72  can be movable along guide rods  76  or any other suitable structure in order to adjust an offset distance of offset camera  72 . The offset distance can be adjusted based on the calculated sharpness curves for offset focusing camera  72  at different distances from the image-forming conjugate plane  80 , as discussed below in connection with  FIG.  7   . 
     The focusing unit  300  can also include two focusing lenses  24  and  22 . Focusing lens  22  can be placed in the same horizontal optical pathway as primary focusing camera  70  and focusing lens  24  can be placed in the same horizontal optical pathway as offset focusing camera  72 . In some embodiments, focusing lenses  22  and  24  achieve the same focal distance as microscope tube lens  10 , to ensure that sensors  71  and  73  are each in focus when they are positioned on the image-forming conjugate plane  80 . Microscope tube lens  10  can include a lens (not shown) for focusing an image of a specimen on sensor  6 , so that the specimen is in focus when sensor  6  is positioned on an image-forming conjugate plane of automatic focus system  100 . 
     Note that in some embodiments, lenses  22  and  24  can be double convex lenses or any other suitable type lenses. In some embodiments, the focal length of the lenses can be based on the optical characteristics of the microscope. 
     As also shown in  FIG.  3   , focusing unit  300  can also include a cutoff dichroic  15  that is positioned above vertical illuminator  13  in the optical pathway of the light reflected off a specimen. The dichroic  15  is positioned so that the light reflected off the specimen that is below the cutoff of the dichroic is reflected at an angle of 90° towards primary focusing camera  70 . A dichroic having a specific cut-off wavelength can be selected in order to reflect the wavelengths of light emitted by secondary illumination source  40  (the “focusing beam”). For example, if the focusing beam is in the range of 400 to 450 nm, then a 450 nm cut-off filter can be used with focusing unit  300  in order to reflect the focusing beam towards primary focusing camera  70 . 
     In some embodiments, focusing unit  300  can include a beam splitter  26  that can be positioned between dichroic  15  and primary focusing camera  70 . The beam splitter  26  can be, for example, a 50/50 beam splitter designed to send 50% of the focusing light beam to primary focusing camera  70  and 50% of the focusing light beam to offset focusing camera  72 . A mirror  28  can be placed at a distance directly above beam splitter  26  and can be designed to direct the beam of light from beam splitter  26  to offset focusing camera  72 . 
     In some embodiments, a cut-off filter  17  can be positioned between dichroic  15  and beam splitter  26  to filter out any light coming from primary illumination source  65  (the “imaging beam”). For example, if imaging beam has a wavelength in the range of 450 nm and above, then a 450 nm cutoff filter can be used to filter out the imaging beam and prevent the imaging beam from transmitting light to focusing cameras  70  and  72 . In other embodiments, two cut-off filters can be used and each filter can be placed, for example, in front of or behind lenses  22  and  24 . 
     Note that, in some embodiments any suitable dichroic, focusing camera, focusing lens, mirror, image sensor, beam splitter and cut-off filter can be used with focusing unit  300 . In some embodiments, any suitable arrangement of these components can be used with focusing unit  300 . The components of focusing unit  300  can be mounted to guide rods or any other suitable structure for connecting the components. Further, in some embodiments, primary focusing camera  70  is not necessary and the focusing operations described herein for primary focusing camera  70 , can instead be performed by imaging camera  5 . 
       FIG.  4    shows example optical pathways, represented by a pair of dashed lines, for automatic focus system  100 , in accordance with some embodiments of the disclosed subject matter. Automatic focus system  100  can be configured so that the light emitted from secondary illumination source  40  (the “focusing beam (FB),” as represented by the shorter dashed lines) is projected onto specimen S and then reflected to focusing cameras  70  and  72 . Autofocus system  100  can also be configured so that light emitted from primary illumination source  65  (the “imaging beam (IB),” as represented by the longer dashed lines) is projected onto specimen S and then reflected to imaging camera  5 . 
     More specifically, in some embodiments, the focusing beam can travel from illumination source  40  through focusing pattern  55  to dichroic  60 . Dichroic  60  can reflect the focusing beam towards vertical illuminator  13 . 
     The imaging beam can travel from primary illumination source  65 , pass through dichroic  60  to combine with the focusing beam. 
     The combined beam can then travel through vertical illuminator  13  to prism  20 . Prism  20  can reflect the light coming from the illumination sources at 90° downwards through a nosepiece and objective  25  to a specimen S. Specimen S can reflect the combined beam upwards through objective  25 , which is then transmitted through prism  20  towards dichroic  15 . Dichroic  15  can separate the transmitted beam back into the imaging beam and focusing beam by, for example, reflecting the wavelengths of the focusing beam towards focusing cameras  70  and  72  and by allowing the wavelengths of the imaging beam to pass through towards camera  5 . 
     In some embodiments, the focusing beam that is reflected by dichroic  15  can pass through cutoff filter  17  to remove any light above the cutoff wavelength. The focusing beam can then travel to beam splitter  26 . Beam splitter  26  can send 50% of the focusing beam towards primary focusing camera  70  by directing the light through focusing lens  22  located in focusing housing  18 . From there the focusing beam, can travel to a light sensor  71  ( FIG.  3   ) in camera  70 . The other 50% of the focusing beam can be directed by beam splitter  26  upwards towards mirror  28 . Mirror  28  can reflect the focusing beam towards focusing lens  24  locating in focusing housing  19 . From there the focusing beam can be directed to sensor  73  ( FIG.  3   ) in offset camera  72 . 
     In some embodiments, the imaging beam that passes through dichroic  15  can pass through an optical filter  11  (e.g., a filter that transmits only the wavelengths from the imaging beam), up through tube lens  10 , and to camera sensor  6  located in imaging camera  5 . 
     In some embodiments, primary focusing camera  70  can be used to determine the in-focus point of a specimen. The focus of a specimen can be adjusted, for example, by moving the objective and stage closer together or farther apart along a Z axis (as shown in  FIG.  1   ). More particularly, primary focusing camera  70  can be used to obtain an image of a specimen at two or more Z positions (e.g., by moving stage  30  and/or objective  25  in a Z direction). From the resulting images, a relative sharpness value can be calculated by control system  108  for each Z position of the specimen to determine the quality of focus. Automatic focus system  100  can use any suitable sharpness equation to calculate the relative sharpness of the resulting images. One example equation that can be used by automatic focus system  100  to calculate a relative sharpness score is a measure of image variance V, normalized by the meanμ to account for intensity fluctuations: 
             V   =       1   μ     ⁢       ∑     i   =   1     N     ⁢       ∑     j   =   1     M     ⁢       [       s   ⁡     (     i   ,   j     )       -   μ     ]     2                 
where s(i,j) is the grayscale pixel value at coordinates (i,j) and N and M represent the number of pixels in the i and j directions respectively. Other example methods for calculating a relative sharpness value that can be used by automatic focus system  100  are described by Sivash Yazdanfar et al., “Simple and Robust Image-Based Autofocusing for Digital Microscopy,” Optics Express Vol. 16, No. 12, 8670 (2008), which is hereby incorporated by reference herein in its entirety. The above disclosed methods are just examples and are not intended to be limiting.
 
       FIG.  5    shows a graph comprising an X axis that represents the relative position of a specimen in a Z direction (the “Z position”) and a Y axis representing a relative sharpness score. The relative Z position represents the distance between the top of stage  30  and the objective  25 . The Z position can be changed either by adjusting a stage  30  towards or away from objective  25  and/or by adjusting objective  25  towards or away from stage  30 . The sharpness curve shown in  FIG.  5    compares, at each measurement point along the curve, the relative sharpness of an image captured by primary focusing camera  70  with the relative Z position. As shown in  FIG.  5   , the sharpness value for a specimen can have a largest measured sharpness (e.g., sharpness score of 70 in  FIG.  5   ) at a given relative position (e.g., Z position  130 ) (that can be referred to as the in-focus position) and may decrease symmetrically on each side of the in-focus position (e.g., Z position  130 ). In some instances, the slope of the curve in  FIG.  5    at the in-focus position can be zero or close to zero. It should be understood that the term “in focus” as used herein is intended to denote when the relative positioning of the objective and the stage are such that a sharpness measurement is at a point at or near the top of a sharpness curve. The term “in focus” is not intended to be limited to perfect or optimal focus. 
     The range of coarse Z movement is represented by lines  137  (e.g., at 500 um) and  142  (e.g., at 2500 μm). The range of fine focus Z movement is represented by lines  136  (e.g., at 1400 μm) and  141  (e.g., at 1600 μm). Note, that the range of Z movement refers to a practical range of movement to achieve different Z positions between objective  25  and stage  30 . The range of Z movement also refers to the range of Z movement where a sharpness calculation can be used to focus a specimen. Arrow  135  shows the sharpness score increasing to a maximum point at Z position  130  (indicating that the image is considered to be in focus as described above) as stage  30  and objective  25  move farther apart and arrow  140  shows the sharpness score decrease from maximum point at Z position  130  as stage  30  and objective  25  continue to move farther apart. 
       FIG.  6    shows an example sharpness curve for offset focusing camera  72 . Similar to  FIG.  5   , the X axis of the graph represents the relative Z position, the Y axis represents a relative sharpness score and line  130  indicates the Z position where the maximum measured sharpness value for primary focusing camera  70  is found. The sharpness curve shown in  FIG.  6    compares, at each point along the curve, the relative sharpness of an image captured by offset focusing camera  72  with the relative Z position. In some embodiments, automatic focus system  100  can use the same equation to calculate the sharpness curve for primary focusing camera  70  and offset focusing camera  72 . 
     As shown in the example of  FIG.  6   , at the in-focus position (e.g., Z position  130 ) determined using primary focusing camera  70  as described above in connection with  FIG.  5   , the relative sharpness value for an image captured by offset focusing camera  72  is around 28 (as indicated by arrow  138 ). This value (e.g., 28) can be stored by control system  108  as the sharpness setpoint for offset camera  72  for that particular specimen, specimen class and/or any other suitable classification group. In some embodiments, a specimen class can be defined based on specimens made from materials of similar reflective qualities. Some example specimen classes can include, but are not limited to: a bare silicon wafer; a semiconductor wafer with a known pattern; and a biological specimen of the same known substances prepared consistently with a glass slide and cover slip. 
     As shown in  FIG.  6   , the sharpness curve for the images taken by offset focusing camera  72  is constantly increasing (as represented by arrow  150 ) between lines  137  and  142  (representing the range of Z movement). Once the sharpness setpoint for a specimen or a class of specimens is found, then offset focusing camera  72  can be used to determine whether to move stage  30  and objective  25  closer together or farther apart. For example, if the sharpness setpoint of a specimen is determined to be 28, and stage  30  is translated in an X/Y plane perpendicular to the Z axis such that the specimen is no longer in focus, then the sharpness value of an image captured by the offset focusing camera  72  can be used, together with the sharpness setpoint and the sharpness curve, to bring the specimen back in focus. For example, if the sharpness setpoint for the image of the specimen, as captured by offset focusing camera  72 , is 28 as described above and a relative sharpness value of an image of the specimen, post stage translation, as captured by offset focusing camera  72  is 52 (for example), then, as evident from the sharpness curve in  FIG.  6   , the distance between the stage and objective must be decreased (e.g., from 2000 μm to 1500 μm) to bring the specimen back into focus. On the other hand, if the relative sharpness value for the image of the specimen as captured by offset focusing camera  72  is less than 28 (e.g., at  20 ), then, as evident from the sharpness curve in  FIG.  6   , the distance between the stage and objective must be increased (e.g., from 1000 μm to 1500 μm) to bring the specimen back into focus. Since the relative sharpness curve of offset focusing camera  72 , together with the sharpness setpoint, indicates whether the distance between the stage and objective must be decreased or increased, fewer images of the specimen can be taken to bring a specimen back into focus. 
     This same information about whether to move the stage and objective closer together or farther apart cannot be gleaned from the sharpness curve shown in  FIG.  5   . For example, as shown in  FIG.  5   , if the largest measured sharpness value of a specimen, as captured by primary focusing camera  70 , is 70, and the actual sharpness value of the specimen is measured to be 51, then the sharpness curve shows that the relative Z position can be either to the right or the left of the in-focus point at Z position  130 . Because the relative Z position can be either to the right or the left of the in-focus point at Z position  130 , the sharpness curve cannot be used to determine whether to move the stage and objective closer together or farther apart. 
       FIG.  7    shows different example sharpness curves (i.e., sharpness curves A, B and C) for offset focusing camera  72  when it is positioned at different distances from image-forming conjugate plane  80  (as shown in  FIG.  3   ). Similar to  FIG.  6   , the X axis of the graph in  FIG.  7    represents the relative Z position, the Y axis represents a relative sharpness score and line  130  indicates the Z position where the maximum measured sharpness value for primary focusing camera  70  is found. Sharpness curves A, B and C compare, at each point along the curves, the relative sharpness of an image captured by offset focusing camera  72  with the relative Z position. Sharpness curve D compares, at each point along the curve, the relative sharpness of an image captured by primary focusing camera  70  with the relative Z position. In some embodiments, automatic focus system  100  uses the same sharpness equation to calculate sharpness curves A, B, C and D. Note that the positioning of offset focusing camera  72  herein refers to the positioning of sensor  73  in focusing camera  72 . 
     Out of the three sharpness curves, curve C represents the sharpness curve for offset focusing camera  72  when it is closest to image-forming conjugate plane  80 . Curve B represents a sharpness curve for offset focusing camera  72  when the offset camera is at a distance farther than its offset distance for curve C, but closer than its offset distance for curve A. Curve A represents a sharpness curve for offset focusing camera  72  when the offset camera is farthest away from image-forming conjugate plane  80 . The offset distance refers to the distance between sensor  73  of offset focusing camera  72  and image-forming conjugate plane  80 . 
     Lines A′, B′ and C′ represent the slopes of the respective curves A, B and C at the Z position (e.g., line  130 ) where the specimen is considered to be in focus (as described above in connection with  FIG.  5   ) for primary focusing camera  70 . The slopes of the curves become steeper as offset focusing camera  72  moves closer to image-forming conjugate plane  80 . A steeper slope, represents a larger change in sharpness versus a smaller change in Z height (also referred to as greater resolution). A steeper slope is desirable because it allows for finer focal adjustment and control. 
     In some embodiments, the range of Z movement necessary to bring a specimen in focus can determine the offset distance. The range of Z movement can be based on, for example: the thickness and/or any other suitable characteristic of a specimen; the specimen class and/or any other suitable grouping of the specimen; and/or the optical characteristics of the microscope (e.g., the magnification of the objective). The range of Z movement can also be chosen to encompass a wide range of specimen types to prevent having to constantly adjust the offset distance. 
     In some embodiments, to determine the appropriate offset distance for automatic focus system  100 , offset focusing camera  72  can be positioned at different offset distances. A sharpness curve can be calculated at each offset distance. The offset distance that produces a sharpness curve that represents a desired range of Z movement and is constantly increasing (represented by a positive slope) or decreasing (represented by a negative slope) in that range, can be selected. 
     More specifically, if the range of Z movement is large (e.g., the distance between lines  137  and  142  shown in  FIG.  7   ), then offset focusing camera  72  can be positioned farther away from image-forming conjugate plane  80  (as represented by curve A). For example, if the desired range of Z movement is between 500 μm and 2500 μm (as represented by lines  137  and  142 ), then the offset camera should not be positioned at the offset distance that produced curve B or curve C, because curves B and curves C both increase and decrease in the desired range of Z movement and cannot be used to determine whether to move the specimen and objective closer together or farther apart. 
     In some embodiments, the offset distance can also be based on the steepness of the sharpness curve at the position where the specimen is in optimum focus for primary focusing camera  70  (e.g., as represented by lines A′, B′ and C′). For example, if the range of Z movement necessary to bring a specimen in focus is small (e.g., between 1300 μm-1700 μm), then offset focusing camera  72  can be placed closer to image-forming conjugate plane  80  (e.g., at the offset distance that produced curve C). Even though a larger offset distance can produce acceptable sharpness curves (e.g., curves B and A), the position of offset focusing camera  72  that produces curve C can be selected because that position has the steepest slope and greatest resolution compared to an offset distance farther away from image-forming conjugate plane  80 . In some embodiments, a position farther away from the image-forming conjugate plane  80  can be selected to accommodate a maximum range of Z movement for automatic focus system  100 , so that the offset focusing camera  72  does not have to constantly be repositioned for specimens of varying thicknesses. 
     Note that offset focusing camera  72  can be positioned at an offset distance to the right or to the left of image-forming conjugate plane  80 . The sharpness slope over the range of Z movement when positioned to the right or to the left will be moving in one direction and opposite of each other. For example, if offset focusing camera  72  is positioned to the right of image-forming conjugate plane  80  and the sharpness slope is increasing over the range of increasing Z values, then if the offset focusing camera  72  is positioned to the left of image-forming conjugate plane  80 , the sharpness slope will be the opposite (i.e., decreasing over the range of increasing Z values). In other words, the sign of the sharpness slope (i.e., whether the slope is positive or negative) depends on whether the offset focusing camera is to the right or left of the image forming conjugate plane. Therefore, if the relative position of the offset focusing camera (i.e., whether the offset focusing camera is to the right or left of the image forming conjugate plane), the sharpness setting, and a sharpness value are known, then whether to increase or decrease the relative positioning of the objective and the stage to achieve better focus can be inferred. 
     In some embodiments, the offset distance for offset focusing camera  72  can be set once for automatic focus system  100 . In other embodiments, the offset distance can vary to accommodate different objectives, different specimen thicknesses, different specimen classes or any other suitable criteria. For example, offset camera  72  can be moved closer to image-forming conjugate plane for higher magnification objectives to accommodate a smaller depth of field (focus) and smaller range of Z movement. In some embodiments the offset distance can be saved by control system  108  as an offset distance setpoint. The offset distance setpoint can be associated, for example, with the thickness and/or any other suitable characteristic of a specimen, the specimen class and/or any other suitable grouping of the specimen, and/or the optical characteristics of the microscope (e.g., the magnification of an objective). The offset distance setpoint can be used to automatically position offset focusing camera  72 . 
       FIG.  8   , with further reference to  FIGS.  1 - 7   , shows at a high level, an example of an automatic focus operation of automatic focus system  100 , in accordance with some embodiments of the disclosed subject matter. Automatic focus process  800  can use automatic focus system  100 . 
     At  810 , a specimen can be placed on stage  30 . 
     If automatic focus system  100  does not know a sharpness setpoint for the specimen (e.g., the value can be obtained by user input or a prior value stored by control system  108  and associated with a particular specimen, a particular specimen class and/or any other suitable classification group for the specimen), then at  820  control system  108  can move stage  30  and objective  25  closer together and/or farther apart until the control system determines, using a suitable sharpness algorithm (as discussed above in connection with  FIG.  5   ), that the images captured by primary focusing camera  70  are in focus (e.g., control system  108  determines the in-focus position (e.g., at Z position  130 , i.e., the Z position when the specimen is considered to be in focus for primary focusing camera  70  as shown in  FIG.  5   ). In some embodiments, imaging camera  5  can be used, instead of primary focusing camera  70 , to determine the largest measured sharpness value for a specimen. In some embodiments, a sharpness curve can be calculated for offset focusing camera  72  based on images captured by the offset focusing camera  72  at the various Z positions of the stage and objective during the focusing process. The sharpness curve for offset focusing camera  72  can be stored as a sharpness curve setpoint and associated with a particular specimen, a particular specimen class and/or any other suitable classification group for the specimen. 
     At  830 , in some embodiments, once the specimen is determined to be in focus, an in-focus image can be captured by imaging camera  5 . 
     At  840 , once the specimen is determined to be in focus by primary focusing camera  70  (or imaging camera  5 ), an image of the specimen can be captured by offset focusing camera  72 . A sharpness value for the captured imaged can be calculated (e.g., using the same sharpness equation used for the primary focusing camera  70 ) and stored by control system  108 . The stored value can be stored as the in-focus sharpness setpoint and associated with a particular specimen, a particular specimen class and/or any other suitable classification group for the specimen. In some embodiments, when the specimen is in focus for primary camera  70  or imaging camera  5 , the absolute position of: stage  30 ; objective  25 ; the top of the specimen on stage  30 ; and/or the distance between the top of stage  30  and objective  25 , can be stored by control system  108  as a position setpoint. The position setpoint can be associated with a particular specimen, a particular specimen class and/or any other suitable classification group for the specimen. 
     At  850 , stage  30  can be moved in an X/Y plane perpendicular to the Z axis. 
     At  860 , in some embodiments, offset camera  72  can be used to capture an image of the specimen at the new X, Y position of stage  30  and control system  108  can calculate the sharpness value for that image. Based on the sharpness value of the image compared to the in-focus sharpness setpoint, control system  108  can determine whether the specimen is in focus at stage  30 &#39;s new X, Y coordinates or the Z height needs to be adjusted, so that the specimen is brought back in focus. For example, based on the sharpness curve shown in  FIG.  6   , if the calculated sharpness value is greater than the stored in-focus sharpness setpoint, then stage  30  and objective  25  can be brought closer together in a Z direction until the sharpness value of an image captured by offset camera  72  is calculated to be the same as the stored sharpness setpoint. Conversely, if the calculated sharpness value is less than the stored in-focus sharpness setpoint, then the stage and objective can be brought farther apart in a Z direction until the sharpness value of an image captured by offset camera  72  is calculated to be the same as the stored in-focus sharpness setpoint. The direction to adjust the Z position can be determined either from a sharpness curve captured for offset camera  72  during the focusing process at  820  or based on the position of offset focusing camera  72  in relation to the image-forming conjugate plane  72 . This process for calculating the sharpness value of an image by the offset focusing camera and comparing it to a stored in-focus sharpness setpoint can be repeated each time the X,Y coordinates of stage  30  changes. 
     At  870 , a new specimen can be placed on stage  30 . If control system  108  determines that there is a sharpness setpoint already associated with the specimen, the specimen class and/or any other suitable classification group for the specimen, then the control system can use images captured by offset focusing camera  72 , as described at  840 , to determine when the new specimen is in focus. For example, an image of the new specimen can be captured by offset focusing camera  72  and the sharpness value can be compared with the sharpness setpoint associated with the new specimen. The stage and objective can be brought closer together or farther apart in a Z direction until the sharpness value of an image captured by offset camera  72  is calculated to be the same as the stored sharpness setpoint. 
     In some embodiments, once the calculated sharpness value for a specimen, using offset focusing camera  72 , corresponds to the sharpness setpoint, primary focusing camera  70  can be used to fine tune the focus of the specimen and the sharpness setpoint of offset focusing camera  72 . For example, using primary focusing camera  70 , sharpness values can be calculated for at least two relative Z positions of the stage and objective to determine whether an estimated maximum sharpness has been achieved or the relative Z-position needs to be adjusted to achieve an estimated maximum sharpness (i.e., the point on the sharpness curve where the slope is 0 or close to 0). Once an estimated maximum sharpness is achieved, the sharpness value of the specimen, using offset focusing camera, can be calculated and stored as the new sharpness setpoint. 
     In some embodiments, control system  108  can also determine whether there is a position setpoint associated with the new specimen, specimen class and/or any other suitable classification group for the specimen, and can position autofocus system  100  at that position setpoint before it begins the aforementioned focusing process. Knowing the relative Z position, reduces the relative Z distance that is needed to focus the specimen and allows the offset camera to be positioned closer to the image-forming conjugate plane. As discussed above in connection with  FIG.  7   , the slope of the sharpness curve can become steeper as offset focusing camera  72  moves closer to the image-forming conjugate plane. A steeper slope, represents greater resolution or a larger change in sharpness versus a smaller change in Z height. A steeper slope can allows for finer focal adjustment and control. 
     The division of when the particular portions of process  800  are performed can vary, and no division or a different division is within the scope of the subject matter disclosed herein. Note that, in some embodiments, blocks of process  800  can be performed at any suitable times. It should be understood that at least some of the portions of process  800  described herein can be performed in any order or sequence not limited to the order and sequence shown in and described in the  FIG.  8    in some embodiments. Also, some of the portions of process  800  described herein can be or performed substantially simultaneously where appropriate or in parallel in some embodiments. Additionally or alternatively, some portions of process  800  can be omitted in some embodiments. 
     Process  800  can be implemented in any suitable hardware and/or software. For example, in some embodiments, process  800  can be implemented in control system  108 . 
     The automatic microscopic focus system and method have been described in detail with specific reference to these illustrated embodiments. It will be apparent, however, that various modifications and changes can be made within the spirit and scope of the disclosure as described in the foregoing specification, and such modifications and changes are to be considered equivalents and part of this disclosure. The scope of the invention is limited only by the claims that follow.