Patent ID: 12222345

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the invention is not intended to limit the invention to these preferred embodiments, but rather to enable any person skilled in the art to make and use this invention.

1. System

As shown inFIGS.1A-1D, an embodiment of a system100for detecting features of a biological sample at an imaging substrate comprises: an illumination module110configured to illuminate a target object (e.g., captured cells of interest within a microfluidic cell capture device) of the biological sample; a platform130configured to position the target object in relation to the illumination module110; a filter module140configured to filter light transmitted to the target object and/or to filter light received from the target object; an optical sensor150configured to receive light from the target object and to generate image data; and a focusing and optics module160configured to manipulate light transmitted to the optical sensor150. The system100can further comprise a control system170configured to control at least one of the illumination module110, the platform130, the focusing and optics module160, the filter module140, and the optical sensor150; a tag identifying system180configured to identify and communicate tag information from system100elements; a thermal control module190configured to adjust temperature parameters of the system100; an image stabilization module200; a processor220configured to process information captured from the target object; and a linking interface230configured to transmit information between the processor220, the optical sensor150, the control system170, and/or the thermal control module190. The system100functions to facilitate manipulation and imaging of biological samples comprising captured cells of interest, in order to enable analyses of captured cells. The system100is preferably configured to receive a microfluidic cell capture device, such as the device described in U.S. application Ser. No. 13/557,510, entitled “Cell Capture System and Method of Use” and/or the device described in U.S. application Ser. No. 14/163,153, entitled “System and Method for Capturing and Analyzing Cells”, which are both incorporated in their entirety herein by this reference. The system100can additionally accept other imaging substrates350, such as microscope slides, tissue processing slides, microarray slides, tissue microarray slides, cell culture plates, and/or any other suitable imaging substrates350. The system100can be capable of providing auto-focusing before image capture, but can alternatively take a series of images at multiple focal lengths, use image post-processing to sharpen the image, or utilize any other suitable method to achieve a focused image of a biological sample.

In a specific embodiment, the system100is configured to image captured cells within a microfluidic cell capture device that captures and isolates single cells of interest. In the specific embodiment, the system100provides unbroken, focused images of all microfluidic cell capture chambers in the microfluidic cell capture device, couples image data with target cell/device identifying information (e.g., location, time) and system parameter information (e.g., illumination information, temperature information), and facilitates light-based cellular diagnostic assays including assays involving fluorescent dyes (e.g., Hoechst dye, Alex Fluor 633, Hex, Rox, Cy5, and Cy5.5). The specific embodiment is further configured to be a benchtop system that operates below a specified decibel level, and is configured to not require room external room darkening to facilitate analyses of captured cells and/or other biological samples. Other variations can involve any other suitable configuration and/or combination of elements that enables imaging of captured cells, and can include elements described in U.S. application Ser. No. 13/557,510, entitled “Cell Capture System and Method of Use”.

1.1 System—Illumination Module

The illumination module110comprises a first illumination subsystem111, and functions to transmit light toward one or more target objects (e.g., captured cells of interest) at the platform130to facilitate analyses of the target object(s). Preferably, the illumination module110comprises a first illumination subsystem111and a second illumination subsystem121, such that multiple types of light-based analyses can be enabled by the system100. The illumination module110can, however, comprise a single illumination subsystem or more than two illumination subsystems to facilitate multiple types of light-based analyses. Additionally, the illumination module110can comprise elements (e.g., housings, filters) configured to reduce or eliminate light not originating from the illumination module110(e.g., light within a room containing the system).

In a first variation, the first illumination subsystem111is a bright-field subsystem111and the second illumination subsystem is a fluorescence subsystem121. The bright-field subsystem111preferably comprises a wide-spectrum light source as a first light source112(e.g., white light source) with an adjustable intensity, and is configured to transmit light through a first set of optics113toward a platform130configured to position captured cells. In other variations, the first light source112may not comprise a wide-spectrum of wavelengths, and/or may not be configured with an adjustable intensity. In one variation, the first light source112comprises a white light emitting diode (LED); however, the first light source112can additionally or alternatively comprise any other light source configured to provide bright-field images. Light from the first light source112thus illuminates a sample at an imaging substrate350at the platform130, and contrast is provided by differential absorbance of light within the sample. The bright-field subsystem111preferably provides true bright-field images, but can additionally or alternatively provide composite bright-field images. The first set of optics113can comprise a collimator, which functions to collimate light from the first light source112, and/or a focusing lens, which functions to focus light from the light source onto a captured cell. The focusing lens can be configured to focus light onto a single object (e.g., captured cell), or can one of a set of focusing lenses configured to focus light onto multiple objects (e.g., captured cells, region of a tissue sample) simultaneously. In a first variation, the first light source112and the first set of optics113are aligned in a vertical direction with respect to a horizontal platform130, such that light is transmitted in a substantially perpendicular direction toward captured cells of interest at the horizontal platform130. As such, in the first variation, the first light source112can be situated inferior to or superior to the platform130. In an example of the first variation, light from the bright-field subsystem111is configured to impinge upon a biological sample comprising cells of interest, wherein the light is transmitted in a direction toward an optical sensor150located above (e.g., superior to) the biological sample, in the orientation shown inFIGS.1B-1D. In another example of the first variation, light from the bright-field subsystem111is configured to impinge upon a biological sample comprising cells of interest, wherein the light is transmitted in a direction toward an optical sensor150located under (e.g., inferior to) the biological sample, in the orientation shown inFIG.2A. In this example, the bright-field subsystem111is further configured to provide consistent illumination in two directions (e.g., in X and Y directions in a two-dimensional plane). However, the first light source112and the first set of optics113can alternatively be configured in any appropriate orientation and configured to direct light (e.g., using a mirror102) to illuminate captured cells of interest with any suitable illumination profile. In other variations, the bright-field subsystem111can only comprise the first light source112and omit the first set of optics113, or can comprise a first set of optics113including alternative or additional elements (e.g., mirror, lens, beam shaper, beam splitter).

In the first variation, the second illumination subsystem121is a fluorescence subsystem121comprising a wide-spectrum light source as a second light source122with an adjustable intensity, preferably including ultraviolet and/or infrared wavelengths of light, and a second set of optics123configured to manipulate light from the second light source122. The fluorescence subsystem121may, however, not be configured to provide an adjustable intensity. In an example, the wide-spectrum second light source122comprises an LED that provides light with wavelengths at least in the range between350-830nm, such that the filter module140can filter light from the second light source122to appropriately enable fluorescence light-based analyses using fluorescent dyes (e.g., Hoechst dye, Alexa Fluor 633, FAM, Hex, Rox, Cy5, Cy5.5). However, the second light source122can additionally or alternatively comprise any other light source(s) configured to facilitate fluorescence light-based analyses. Additionally, the second light source122can comprise multiple light sources (e.g., multiple LEDs). In one example comprising multiple light sources, the multiple light sources can produce a certain range of light wavelengths, such that light from the multiple light sources can be filtered to reduced wavelength ranges for imaging and analysis of target objects according to specific assay protocols. The second set of optics123can comprise a collimator, which functions to collimate light from the second light source122, and/or a focusing lens, which functions to focus light from the light source onto a captured cell. The focusing lens can be configured to focus light onto a single target object (e.g., captured cell), or can be one of a set of focusing lenses configured to focus light onto multiple target objects (e.g., captured cells, region of a tissue sample) simultaneously. In a first variation, the second light source122and the second set of optics123are aligned in a horizontal direction with respect to a horizontal platform130, such that light is transmitted in a substantially parallel direction prior to being reflected (e.g., using a mirror102) toward captured cells of interest or tissue at the horizontal platform130. In an example of the first variation, light from the second illumination subsystem121is configured to impinge upon a biological sample comprising cells of interest, wherein the light from the second illumination subsystem121is transmitted in a direction away from an optical sensor150located above the biological sample, after being reflected by a mirror102and a dichroic mirror143, in the orientation shown inFIGS.1B-1C. In another example of the first variation, light from the fluorescence subsystem121is configured to impinge upon a biological sample comprising cells of interest, wherein the light is transmitted in a direction away from an optical sensor150located under the biological sample, in the orientation shown inFIG.2A. However, the second light source122and the second set of optics123can alternatively be configured in any appropriate orientation to illuminate captured cells of interest with any suitable illumination profile. In other variations, the fluorescence subsystem121can only comprise the second light source122and omit the second set of optics123, or can comprise a second set of optics123including alternative or additional elements (e.g., mirror, lens, beam shaper, beam splitter).

In alternative variations, at least one of the first illumination subsystem111and the second illumination subsystem121can comprise a dark-field subsystem, a confocal subsystem, a phase-contrast subsystem, and/or any other suitable imaging subsystem. Additionally, in other variations, at least one of the first illumination subsystem111and the second illumination subsystem121can be coupled to an actuation subsystem128configured to translate, rotate, or angularly displace a illumination subsystem111,121relative to a biological sample comprising cells of interest.

1.2 System—Platform

As shown inFIGS.1A,1B, and2A, the platform130comprises a platform control module133and a guide138, and can additionally or alternatively include an image normalizer129. The platform130functions to receive and align a cell capture device or other imaging substrate350relative to the illumination module110and/or the optical sensor150, in order to enable light-based analyses of captured cells of interest within the cell capture device or other imaging substrate350. In some variations, the platform130can be automatically controlled by a control system170, in order to facilitate automated functions including autofocusing of objects of interest, self-calibration, cell capture device interrogation, cell capture device agitation, or any other suitable function. In other variations, the platform130can be semi-automatically controlled or manually controlled, such that a user or other entity can manipulate the platform130in some manner (e.g., using knobs or dials mechanically coupled to the platform130). Additionally, the platform130is preferably cleanable (e.g., using ethanol), such that the platform130can be reusable for multiple runs of analyses. The platform130is preferably situated between the first and the second illumination subsystems111,121, as described above, but can be located relative to any other suitable element of the system100in any other suitable manner.

As shown inFIGS.1A,1B,2A, and4B, the platform control module133functions to facilitate motion of the platform130relative to other elements of the system100. The platform control module133preferably enables motion of the platform130in at least one direction, but can additionally be configured to enable motion of the platform130in two or three directions (e.g., X, Y, and/or Z directions). The platform control module133can additionally or alternatively provide rotational motion or any other suitable motion of the platform. To produce linear translations of the platform130, a first variation of the platform control module133can comprise a translation stage334with a translation controller335(e.g., knobs that affect translation, actuator module that affects translation). The translation stage334in the first variation is also coupled to the platform130in order to enable translations of the platform130in X, Y, and/or Z directions. In an example of the first variation, as shown inFIGS.5A-5C and6, a first knob134with a flexible shaft extension can affect a translation of the platform130in the X direction, a second knob135with a flexible shaft extension can affect a translation of the platform130in the Y direction, and a third knob136with a flexible shaft extension can affect a translation of the platform130in the Z direction. Other variations of the platform control module133can comprise any other suitable element or subsystem (e.g., guiderails, springs, lead screws) configured to produce linear translations of the platform130.

As shown inFIG.4B, the platform control module133can further be configured to angularly displace or rotate the platform130, in order to provide images of target objects (e.g., captured cells of interest) in multiple orientations and/or to position target objects relative to other elements of the system100. Angular displacement or rotation of the platform130can further facilitate auto-focusing and/or calibration functions of the system100. As such, the platform control module133can be configured to angularly displace the platform about an axis parallel to the platform130, about an axis perpendicular to the platform130, and/or about an axis oriented in any other suitable manner relative to the platform. In an example, the platform control module133can be configured to angularly displace the platform130at a specified angle about an axis parallel to the platform130, which results in a distribution of focal lengths across the platform (e.g., some platform locations will be in better focus than others based on the different resultant focal lengths). In the example, contrast differences generated from platform locations at different focal lengths are then interrogated by a processor220that determines the location with the greatest contrast, a measure indicative of the optimal focal length. The platform control module133in the example then angularly displaces the platform130to a horizontal configuration (e.g., a non-angularly displaced orientation), and translates the platform130, to achieve the optimal focal length relative other system elements. In another example, the platform control module133displaces the platform about an axis perpendicular to the platform130, such that different objects at the platform (e.g., imaging substrates350) can be rotated into position and processed using the system100.

In automated variations of the system100, the platform control module133can comprise an actuator configured to automatically control motion of the platform130. The actuator is preferably configured to affect motion of the platform130in at least two directions (e.g., X and Y directions); however, the actuator can be configured to affect motion of the platform130in less than two directions, more than two directions (e.g., X, Y, and Z directions), and/or in rotation. In an example of an automated variation, as shown inFIG.1B, the platform control module133can comprise at least one motor coupled to a translation controller (e.g., of a translation stage334), such that an actuation provided by the motor produces a translation of the platform130. Specifically, the motor can be coupled to an X, Y, and/or Z translation stage controller335to produce motion of the platform130. In another example, the platform control module133can comprise a stepper motor or any other suitable actuator, coupled to the platform130, which enables rotation of the platform130and a rotational position of the platform130to be assessed. Other automated variations of the system100can comprise any suitable actuator coupled to any suitable platform translator or rotator to control motion of the platform130.

The guide138functions to receive and align an imaging substrate350that contains a biological sample and/or target objects (e.g., captured cells of interest), such that the biological sample and/or target objects can be properly imaged and analyzed. The guide138can be a suitably-sized recess at one surface of the platform130, and/or can comprise a ridge, rail, or tab configured to align the imaging substrate350in relation to the platform130. Furthermore, the guide138can preferably only receive the imaging substrate350in one orientation, such that positive orientation confirmation is enabled by the guide138; however, the guide138can alternatively be configured to receive an imaging substrate350in multiple orientations. The guide138preferably has at least one aperture in order to enable light transmission through the imaging substrate350, thereby facilitating imaging of a target object at the imaging substrate350. The guide138can additionally be one of a set of guides of the platform130, such that the platform is configured to receive and align multiple imaging substrates350. In one variation, the platform130can include an array of guides arranged in multiple rows, as shown inFIG.3A, and in another variation, the platform130can include one or more guides138in a circular arrangement, as shown inFIG.4A, such that a rotation of the platform130rotates successive imaging substrates350, containing target objects (e.g., captured cells of interest), with respect to other elements of the system100. Preferably, each guide138in the set of guides is identical; however, each guide138in the set of guides can alternatively be non-identical, such that different imaging substrates350(e.g., comprising different morphologies) can be received by the platform130. Additionally or alternatively, the platform130can comprise a single guide138that is adjustable in order to accommodate differently sized imaging substrates350.

As shown inFIGS.3A and4A, the guide138can further include a retainer139that holds the imaging substrate350at a specific location position relative to the rest of the platform130. The retainer139is preferably capable of holding at least one imaging substrate350(e.g., cell capture device, glass slide, cartridge). In one variation, the retainer139can be a clip that biases the imaging substrate350against a brace, a recess in a surface of the platform130, or any other suitable retainer139. The platform130can be configured to accommodate one imaging substrate350at a time with a guide138and/or a retainer139, as shown inFIG.2B, but can alternatively be configured to accommodate multiple imaging substrates350simultaneously with multiple guides and/or multiple retainers, as shown inFIGS.1B,1C,3A and4A.

As shown inFIGS.3C, the image normalizer129is preferably coupled to the platform130and functions to facilitate calibration of the system100. The image normalizer129preferably enables at least one of calibration of exposure and calibration of focus, but can additionally or alternatively enable calibration of other aspects of the system100. Preferably, the image normalizer129is located within the same plane as the target object(s) intended to be imaged/analyzed by the system100, such that a calibration using the image normalizer129can be adapted to facilitate imaging and/or analysis of the target object. The image normalizer129can additionally comprise a surface with features similar to those of target objects (e.g., captured cells of interest from a biological sample), to improve the suitability of the calibration. The image normalizer129can be in a fixed location relative to the platform130, but can alternatively be configured to have an adjustable location relative to the platform130. The image normalizer129can further enable automatic calibration of an aspect of the system100in automated variations of the system100.

In other variations, the platform130additionally include or be coupled to a fluidic manifold127coupled to a fluid source, as shown inFIG.1A, wherein the manifold127interfaces with an inlet and an outlet of a microfluidic cell capture device, such as the one described in U.S. application Ser. No. 13/557,510, entitled “Cell Capture System and Method of Use” or U.S. application Ser. No. 14/163,153, entitled “System and Method for Capturing and Analyzing Cells”. The manifold127can thus enable visualization of real-time flow through the microfluidic cell capture device. In variations of the platform130configured to accommodate multiple imaging substrates350, the manifold127can be configured to interface with inlets and outlets of multiple imaging substrates350(e.g., at openings of the manifold), in order to provide visualization of real-time flow through multiple cell capture devices; however, the manifold127can be configured in any other suitable manner.

In a first specific example, as shown inFIGS.1B and4A, the platform130comprises a guide138configured to receive and retain a microfluidic cell capture device or a glass slide with a 1″×3″ footprint and a thickness between 1 mm and 2 mm. The guide138in the first specific example is one of a set of nine guides arranged in a circular array, as shown inFIG.2D, such that the platform130accommodates up to nine microfluidic cell capture devices or other imaging substrates350. In the first specific example, the platform130is rotatable (with a platform control module133) about an axis perpendicular to the platform130through an angular displacement of at least 180° in clockwise and counterclockwise directions; however, in variations of the first specific example, the platform130can be rotatable through any other suitable angular displacement (e.g., 360° in one or two directions, less than 360° in one direction, etc.). In the first specific example, the platform control module133can additionally translate the platform in an X direction by a span of 9″ and in a Y direction by a span of 5″, using a multi-axis (e.g., X-Y) actuation system and a set of guide rails coupled to the platform. Thus, the first specific example allows each of up to nine microfluidic cell capture device(s)/glass slide(s) to be individually imaged and analyzed by the first specific example of the system100. In other variations, the platform130can, however, comprise any suitable combination of elements and/or variations described to facilitate reception and alignment of an imaging substrate350relative to the illumination module110and/or the optical sensor150.

In a second specific example, as shown inFIGS.2B and3A, the platform130comprises a guide138configured to receive and retain a microfluidic cell capture device or a glass slide with a 1″×3″ footprint and a thickness between 1 mm and 2 mm. The guide138in the second specific example is one of a set of guides arranged in a 2×4 array, as shown inFIG.2A, such that the platform130accommodates up to eight microfluidic cell capture devices or glass slides. In the second specific example, the platform130has a footprint of 9″×5″ and is translatable (with a platform control module) in an X direction by a span of 9″ and in a Y direction by a span of 5″. Thus, the second specific example allows each of up to eight microfluidic cell capture device(s)/glass slide(s) to be individually imaged and analyzed by the second specific example of the system100. In other variations, the platform130can, however, comprise any suitable combination of elements and/or variations described to facilitate reception and alignment of an imaging substrate350relative to the illumination module110and/or the optical sensor150.

1.3 System—Filter Module

The filter module140comprises an excitation filter141configured to receive light from a fluorescence subsystem121and transmit light at excitation wavelengths, a dichroic mirror142configured to receive and reflect light from the excitation filter141toward target objects at the platform130, and an emission filter143configured to receive and transmit light from the target objects toward an optical sensor150. The filter module140thus functions to transmit light at excitation wavelengths toward target objects (e.g., captured cells of interest) and to receive light at emission wavelengths from the target objects, in order to facilitate imaging and analysis of the target objects. The filter module140is preferably one of a set of filter modules of the system loo; however, the system100can alternatively include only a single filter module. The filter module(s)140can comprise a set of excitation filters144, a set of emission filters145, and a set of dichroic mirrors146, such that multiple ranges of excitation light can be transmitted, and multiple ranges of emitted light can be transmitted to the optical sensor150. In variations comprising a set of excitation filters141, the set of excitation filters141can include band pass filters configured to transmit light between two bounding wavelengths, short pass filters configured to transmit light below a certain wavelength, and long pass filters configured to transmit light above a certain wavelength. Additionally, the set of excitation filters141can comprise interchangeable filters, such that individual excitation filters can be interchanged to provide different excitation wavelengths of light, and multiple excitation filters can be stacked to provide composite analyses; however, the set of excitation filters141can alternatively be fixed, such that the filter module140is only configured to transmit a fixed range of excitation wavelengths.

In a first variation comprising a set of excitation filters144, excitation filters141in the set of excitation filters144are chosen to transmit different desired ranges of excitation wavelengths. In a first example of the first variation, the set of excitation filters144can comprise a filter that transmits light at wavelengths from 350-390 nm (for Hoescht dye-based assays), a filter that transmits light at wavelengths from 420-480 nm (for other Hoescht dye-based assays), a filter that transmits light at a nominal wavelength of 632 nm (for Alexa Fluor 633-based assays), and a filter that transmits light at a nominal wavelength of 647 nm (for other Alexa Fluor 633-based assays). In a second example of the first variation, the set of excitation filters144can comprise a filter that transmits light at wavelengths from 450-490 nm (for FAM-based assays), a filter that transmits light at wavelengths from 510-540 nm (for Hex-based assays), a filter that transmits light at wavelengths from 555-600 nm (for Rox-based assays), a filter that transmits light at wavelengths from 615-635 nm (for Cy5-based assays), and a filter that transmits light at wavelengths fro 665-685 nm (for Cy5.5-based assays).

The dichroic mirror142of the filter module140is configured to align with an excitation filter141, and functions to receive and reflect light from the excitation filter141toward a target object at the platform130. The dichroic mirror142also functions to receive and transmit light from an emission filter143toward an optical sensor150, which is described in more detail below. In variations comprising a set of dichroic mirrors145, each dichroic mirror142in the set of dichroic mirrors145is preferably identical in orientation relative to an excitation filter141or a set of excitation filters144, and an emission filter143of a set of emission filters146. The dichroic mirror142or the set of dichroic mirrors145can also be configured to reflect and transmit appropriate wavelengths of light based on the application.

The emission filter143is configured to align with a dichroic mirror142, and functions to transmit emission wavelengths of light from the target object at the platform130, and to filter out excitation wavelengths of light. The filter module140can further comprise a set of emission filters146, such that multiple different ranges of light wavelengths can be detected from the target objects at the platform130. In variations comprising a set of emission filters146, the set of emission filters143can include band pass filters, configured to transmit light between two bounding wavelengths, short pass filters configured to transmit light below a certain wavelength, and long pass filters configured to transmit light above a certain wavelength. Preferably, the set of emission filters146is interchangeable and/or stackable, such that individual emission filters can be interchanged or stacked to transmit and/or block different wavelengths of light; however, the set of emission filters146can alternatively be fixed, such that the filter module140is only configured to transmit a fixed range of emission wavelengths.

In a first variation comprising a set of emission filters146, emission filters143in the set of emission filters146are chosen to transmit different desired ranges of emission wavelengths. In an example of the first variation, the set of emission filters146can comprise a filter that transmits light at wavelengths from 507-540 nm (for FAM-based assays), a filter that transmits light at wavelengths from 557-580 nm (for Hex-based assays), a filter that transmits light at wavelengths from 618-638 nm (for Rox-based assays), a filter that transmits light at wavelengths from 655-680 nm (for Cy5-based assays), and a filter that transmits light at wavelengths from 700-830 nm (for Cy5.5-based assays).

The filter module140can be fixed within the system100, but can alternatively be coupled to an actuator configured to displace and/or align the filter module140relative to other system elements. As such, the filter module140can be coupled to a filter stage149coupled to the actuator and configured to translate and/or rotate the filter module140into position with respect to one or more light sources112,122of illumination subsystems111,121of the illumination module110. Furthermore, the filter module140can be one of a set of filter modules coupled to a filter stage149, such that each filter module140in the set of filter modules140can be translated or rotated into position with respect to one or more light sources112,122of illumination subsystems111,121of the illumination module110. As such, the filter stage149preferably includes at least one aperture configured to allow light to be transmitted through the filter module(s)140to a target object at the platform130; however, the filter stage149can additionally or alternatively be substantially transparent to allow light transmission, or can allow light transmission in any other suitable manner. Additionally, the filter stage149can be defined by a circular footprint, a rectangular footprint, or any other suitable footprint (e.g., polygonal, non-polygonal). The filter stage149is preferably situated superior to the platform130and inferior to an optical sensor150; however, the filter stage149can alternatively be situated relative to other elements of the system100in any other suitable manner.

In one variation, the filter stage149can be coupled to an actuator that translates the filter stage149and the filter module(s)140along one or more axes (e.g., X, Y, and/or Z axes) into a desired position in a consistent manner (e.g., using a linear encoder, using a sensor able to provide position detection, etc.). In an another variation, the filter stage149can be coupled to an actuator that rotates the filter stage149and the filter module(s)140into a desired position in a consistent manner (e.g., using a rotary encoder, using a stepper motor, etc.), about an axis perpendicular to a planar surface of the filter stage149. In this variation, the filter stage149is preferably rotatable by at least 180° in clockwise and counterclockwise directions; however, in variations of this variation, the filter stage149can be rotatable through any other suitable angular displacement (e.g., 360° in one or two directions, less than 360° in one direction, etc.). The axis of rotation of the filter stage149is preferably offset and parallel to the axis of rotation of the platform130in variations of the system100including a rotating platform130; however, the axis of rotation of the filter stage149can alternatively be non-offset and/or non-parallel to the axis of rotation of the platform130in variations of the system100including a rotating platform130. In still another variation, the filter stage149can be coupled to one or more actuators that translate the filter stage149and the filter module(s)140along one or more axes (e.g., X, Y, and/or Z axes) and rotate the filter stage149and the filter module(s)140into a desired configuration. In an example, as shown inFIG.1A, the filter module140is one of nine filter modules coupled to a filter stage149defining a substantially circular geometry, with apertures defined within the filter stage149to allow light transmission through the apertures. In the example, each filter module140can be rotated into alignment with a second light source122of a second illumination subsystem121(e.g., a fluorescence subsystem), thereby allowing light from the second light source122to be transmitted through at least one excitation filter141of a filter module140, and to be reflected at a 90° angle by a dichroic mirror142toward a target object at the platform130, and allowing light from the target object to be transmitted through an emission filter143of the filter module140toward an optical sensor150. As such, alignment of a filter module140in the example aligns the excitation filter141with the second light source122, and simultaneously aligns the emission filter143with the optical sensor150. However, in variations of the example, the filter module(s)140can be positioned into alignment with any other suitable elements of the system100in any other suitable manner.

In another specific example of the filter module140, in the orientation shown inFIG.2B, the filter module140comprises an excitation filter141oriented perpendicular to an emission filters143, with a dichroic mirrors142bisecting an angle between two planes formed by the faces of the excitation filter141and the emission filter143. In the specific example, light from the excitation filter141is thus substantially reflected at a 90° angle toward the platform130, and light from the emission filter143passes in a substantially straight direction through the dichroic mirror142toward the optical sensor150. Other variations of the filter module140can include any configuration of dichroic mirror(s), excitation filter(s), and/or emission filter(s) that enable transmission of light of excitation wavelengths toward a target object, and transmission of light from the target object toward an optical sensor150.

1.4 System—Optical Sensor and Focusing and Optics Module

The optical sensor150is configured to align with an emission filter143of the filter module140, and functions to receive light from the emission filter143to facilitate imaging and analysis of a target object (e.g., captured cell of interest). Preferably, the optical sensor150is oriented perpendicular to the platform130, as shown inFIGS.1B,2A, and2B, such that light from a target object at the platform130can be transmitted directly toward the optical sensor150. In one variation, the optical sensor150is situated superior to the filter module140and the platform130, and in another variation, the optical sensor is situated inferior to the platform. However, the optical sensor150can be oriented in any suitable configuration relative to the platform130and/or the filter module140. The optical sensor150can comprise a photodiode comprising a photoelectric material configured to convert electromagnetic energy into electrical signals; however, the optical sensor150can alternatively comprise any other suitable appropriate photodetector for facilitating analysis of biological samples. The optical sensor150can comprise a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) sensor, a line scanner, or any other suitable imaging element. Additionally, the optical sensor150can facilitate imaging in color (e.g., red, green, blue, etc.). In an example, the system100comprising the optical sensor150can enable a 3-color analysis of a sample comprising captured cells of interest within 60 minutes. The optical sensor150can further provide image data in any suitable resolution (e.g., 1-10 pixels/micron, 5-100 megapixels) to distinguish between target objects and target object features, and can detect intensities of electromagnetic energy above a suitable threshold and between a certain range of wavelengths (e.g., 420-830 nm). In a specific example, the optical sensor150is configured to enable differentiation of a single cancer cell in a background of contaminating white blood cells by providing images of sufficient resolution and/or color imaging for fluorescent detection. In the specific example, as shown inFIGS.7A and7B, the optical sensor150can further provide a suitable resolution of image data, such that the system100can differentiate between specific cell capture pore locations155(e.g., addresses) of a microfluidic cell capture device, such as that described in U.S. application Ser. No. 13/557,510 or U.S. application Ser. No. 14/163,153. In the specific example shown inFIGS.7A and7B, the pore locations155are characterized by tags translatable to a binary number by a processor220, and include a series of characters wherein a character with a dot indicates a value of “1” and a character without a dot indicates a value of “0”. As such, in the specific example, each tag translatable to a binary number has a series of nine characters, each character having a dot or no dot that is detectable by the optical sensor150and/or a tag identifying system180as described in further detail below. In another variation of this specific example, a combination of dots can be used as a tag wherein a feature (e.g., relative distance, color, intensity, shape, etc.) between the dots indicate a precise location of the tag within the microfluidic device. In variations of the specific example, however, the pore locations155can be characterized by any other suitable tag (e.g., RFID tag, visually detectable tag, non-visually detectable tag, etc.) and be detectable by any other suitable method (e.g., RF sensing, visual detection, etc.).

The focusing and optics module160preferably comprises a lens161configured to focus light from the illumination module onto a target object at the platform130, and/or a lens162configured to focus light from the target object at the platform130onto the optical sensor150. The lens can be any suitable lens (objective lens) with any suitable magnification (e.g., 10×-40×) and numeric aperture (e.g., ¼″). The lens161,162can also be one of a set of lenses configured to focus light onto individual target objects (e.g., individual lenses focus light onto individual captured cells of interest), or can be a single lens161configured to focus light onto multiple target objects (e.g., captured cells of interest within a microfluidic cell capture device, a tissue region, etc.) at the platform130. The lens(es)161,162can be aligned with the excitation filter141, the dichroic mirror142, and/or the emission filter143of the filter module140, such that light transmitted from or reflected off of the excitation filter141, the dichroic mirror142, and/or the emission filter143is appropriately focused. The lens(s) can however, be aligned in any suitable configuration relative to other elements of the system100and configured to focus incident light by way of any suitable number of optics elements (e.g., dichroic mirrors, mirrors, etc.).

The lens(es)161of the focusing and optics module160can be further configured to translate in one or more directions and/or rotate about any suitable number of axes, to facilitate focusing or auto-focusing of light onto the platform130and/or onto the optical sensor150. In variations wherein the lens(es)161of the focusing and optics module160are configured to translate, translation can be facilitated using an optics manipulation module167, including an actuator166and/or a lens selector165, to enable automated or semi-automated functionalities (e.g., autofocusing, automagnification, etc.). The actuator166preferably couples to the lens(es)161,162and/or the lens selector165, and provides translation along at least one axis (e.g., X-axis, Y-Axis, Z-axis); however, the actuator166can be configured to couple to any other suitable element of the system100in order to enable translation of elements of the focusing and optics module160, and/or can provide translation along multiple axes (e.g., X and Z-axes, Y and Z-axes, X and Y-axes). The lens selector165preferably rotates one of a set of lenses into alignment (e.g., as in a revolving nosepiece); however, variations of the lens selector165can additionally or alternatively translate a lens of a set of lenses into alignment.

In a specific example, as shown inFIGS.1C and1D, the optics manipulation module167includes a revolving nosepiece as the lens selector165, configured to reversibly couple to three objective lenses that can be rotated into alignment with a corresponding element of the system100(e.g., a filter module140, a first illumination subsystem111, a second illumination subsystem121, etc.). The revolving nosepiece in the specific example rotates about an axis angularly displaced from a vertical axis, in the orientation shown inFIGS.1C and1D, such that an aligned lens is rotated into a vertical configuration, and a misaligned lens is rotated into a non-vertical configuration. In the specific example, the revolving nosepiece is coupled to a linear translation stage (e.g., ThorLabs MTS25/M-Z8 translation stage) as an actuator166configured to translate the lens selector165along a Z-axis, in the orientation shown inFIGS.1C and1D. In further detail, to provide translation along the Z-axis, an optical shaft168coupled to the revolving nosepiece and concentric with an aligned lens161,162is coupled to the actuator166by an L-shaped plate (e.g., an L-bracket), thereby facilitating motion of the lens161,162along a Z-direction. Variations of the specific example can, however, include an optical shaft168not aligned with a lens161,162of the focusing and optics module160, and/or can include coupling in any other suitable manner to affect translation of a lens161,162along any suitable axis. In the specific example, the actuator166is coupled to a controller configured to provide autofocusing of the focusing and optics module160; however, variations of the specific example can omit coupling between a controller and the actuator166, and enable manual translation of the lens(es)161,162. Variations of the specific example can further allow rotation or translation of the lens(es)161,162of the focusing and optics module160into any other suitable configuration, in any other suitable manner.

Furthermore, while variations and examples of translation and/or rotation in the platform130, the filter module140, and the focusing and optics module160have been described above, other embodiments of the system100can include translation, rotation, and/or relative motion through any suitable path, of any suitable element of the system100, in order to facilitate light transmission and alignment of optics elements in any other suitable manner.

1.6 System—Other Elements

As shown inFIGS.1A and2A, the system100can further comprise a tag identifying system180. The tag identifying system180functions to read barcodes, QR codes and/or any other identifying tags181of the system100, and to communicate information from the identifying tags to a processor220. The tag identifying system180can be coupled to the illumination module110, as shown inFIG.2A, to facilitate identification and reading of tags located on imaging substrates350coupled to the platform130, or any other suitable system element. In other variations, the tag identifying system180may not be coupled to the illumination module110. The tag identifying system180is preferably fixed in location, but can alternatively be configured to move relative to other system elements. In one alternative variation, the tag identifying system180can be a standalone unit that is configured to be manipulated by a user to scan tags or labels located on elements of the system100. The tag identifying system180can comprise a barcode reader, a radio-frequency identification (RFID) reader, a QR code reader, a nearfield communication device, or any other suitable element implementing a mechanism that can identify a unique identifier located on the an imaging substrate350or other aspect of the system100(e.g., glass slide, cartridge, cell capture device, etc.). The tag identifying system180can alternatively or additionally be configured to parse and interpret non-encoded information (e.g., text) on an identifying tag181. In some variations of the system100, the optical sensor150can additionally function as a tag identifying system180.

As shown inFIG.3B, a tag181intended to be identified and/or read by the tag identifying system180preferably communicates information to the tag identifying system180upon being read. The information can comprise information related to imaging substrate350(e.g., cell capture device, glass slide) identification information, protocol information (e.g., staining protocol information), information related to suggested system parameters required to actualize a protocol, information related to calibration of the system100with regard to a specific imaging substrate350, information related to contents of an imaging substrate350, information configured to facilitate positive location identification of an imaging substrate350or locations within an imaging substrate350, and/or any other suitable type of information. The information can be coupled to (e.g., embedded within) image data captured by the optical sensor150, and/or can be communicated to the processor220using any other suitable means.

As shown inFIGS.1A and7, the system100can further comprise a thermal control module190, which functions to controllably heat and/or cool aspects of the system100to facilitate imaging and analysis of target objects (e.g., captured cells of interest). As such, the thermal control module190controls thermal parameters of at least one of the imaging substrate and a biological sample at the imaging substrate. The thermal control module190is preferably coupled to the platform130, but can alternatively be at a location within proximity of the platform130, or may not be within proximity of the platform130. The thermal control module190can be configured to heat aspects of the system by conduction, convection, and/or radiation using a heating element. The thermal control module190can additionally or alternatively comprise a cooling element configured to cool or modulate heat within the system100. Alternatively, cooling can be enabled by deactivating a heating element. The thermal control module190preferably includes electric heaters, but can alternatively include inductive heaters, ceramic heaters, or any other suitable heaters. The thermal control module190can additionally include a heat sink, heat pump, heat exchanger, fan, or any other suitable passive or active cooling mechanism. The thermal control module190is preferably optically transparent to facilitate unobstructed imaging, but can alternatively have any other suitable optical property such that imaging by the system100is not obstructed. In variations, the thermal control module190can be configured to move out of a field of view after heating and/or cooling a substrate, to enable unobstructed imaging.

In one variation, the thermal control module190comprises a single element configured to contact a surface of an imaging substrate350. In another variation, the thermal control module includes multiple elements, wherein each element is configured to heat or cool a given portion of an imaging substrate350. In one example, the thermal control module190can be used to control the temperature of a microfluidic cell capture device being imaged and/or analyzed by the system100, by heating and/or cooling the microfluidic cell capture device according to a specific protocol during imaging. In an example, of the variation, the thermal control module190can heat the microfluidic cell capture device to incubate the cells of interest captured therein, and can cool microfluidic cell capture device to quench a reaction or incubation process.

The system100can further comprise an image stabilization module200configured to reduce or eliminate artifacts within image data due to unwanted system100motion. In one variation, as shown inFIG.8, the image stabilization module200can comprise vibration isolators210(e.g., feet, pads, platforms) configured to reduce or entirely eliminate system vibration. In another variation, the image stabilization module200can comprise image stabilization software, implemented on a processor220configured to receive image data from the optical sensor150. The image stabilization software can be configured to anticipate and counteract system motion (e.g., by moving the platform130, optical sensor150, and/or focusing and optics module160in a compensatory manner). The image stabilization software can alternatively be configured to post-process image data comprising unwanted motion artifacts, in order to remove the unwanted motion artifacts. In other variations, the image stabilization module200can comprise any other suitable image stabilization device or method.

As shown inFIGS.1A and2A, the system100can further comprise a control system170, which functions to control at least one of parameters of the illumination module no (e.g., intensity), motion of the platform130, filter configurations of the filter module140, imaging parameters of the optical sensor150, identification and reading of tags181by the tag identifying system180, temperature parameters provided by the thermal control module190, and/or any other system function. Thus, the control system170can be electronically and/or physically coupled to the illumination module, the platform130, the filter module140, the optical sensor150, the focusing and optics module160, the tag identifying system180, the thermal control module190, and/or the image stabilization module200. The control system170can enable fully-automated control of parameters of the system100, or can facilitate semi-automated/manual control of parameters of the system100.

In a variation wherein the control system170is coupled to the illumination module no, the control system170can function to adjust light intensity provided by the illumination module no. For example, the control system170can control bright field illumination intensity and fluorescence illumination intensity using potentiostats or other suitable elements. In a variation wherein the control system170is coupled to the platform130, the control system170can function to manipulate translation, angular displacement, and/or rotation of the platform130about any suitable number of axes. In a variation wherein the control system170is coupled to the filter module140, the control system170can facilitate adjustments to filter configurations (e.g., interchanging and/or stacking of filters) to enable various light-based biological sample assays to be performed. In a variation wherein the control system170is coupled to the optical sensor150, the control system170can adjust image capture parameters (e.g., resolution, capture, exposure, etc.). In a variation wherein the control system170is coupled to the focusing and optics module160, the control system170can facilitate motion of the platform130and/or the focusing and optics module160, in order to enable autofocusing functions of the system100. For example, the system100can autofocus to depth fiducials of a cell capture device, or can autofocus on individual cells captured within a cell capture device. In a variation wherein the control system170is coupled to the tag identifying system180, the control system170can function to automate reading of tags181, and can further function to facilitate transfer of information from the tags181to a processor220. In a variation wherein the control system170is coupled to a thermal control module190, the control system170can facilitate heating of an imaging substrate350to a specified thermal state (e.g., temperature), maintaining the imaging substrate350at the specified thermal state, and/or cooling the imaging substrate350. Other variations of the control system170can function automate handling, transfer, and/or storage of other elements of the system100. Alternative combinations of the above variations can involve a single control element, or multiple control elements configured to perform all or a subset of the functions described above.

As shown inFIG.1A, the system100can further comprise a processor220, which functions to receive and process information from the optical sensor150, the control system, a tag identifying system180, and/or any other suitable system element. Preferably, the processor220implements image processing software configured to process image data from the optical sensor150, and can be coupled to a user interface211with a display, as shown inFIG.1A. In one such variation, the processor220can include a module configured to receive a dataset from the optical sensor150to calibrate at least one of the optical sensor150and the optics manipulation module167, based upon a distribution of focal lengths between the optical sensor150and the platform130(e.g., based upon a focal length providing a maximum contrast level). In another variation, the processor220can include a module configured to facilitate analysis of real-time fluid flow at the at least one imaging substrate350based upon data generated by the optical sensor150. In another variation, the processor220can include a module configured to translate a series of characters, physically defined at an imaging substrate350(e.g., proximal to a pore of the array of parallel pores) and detectable using the optical sensor150, into a binary number indicative of an address (e.g., of the pore) characterized by the series of characters. The processor220can, however, include any other suitable modules configured to perform any other suitable function.

In variations comprising a user interface211with a display, the user interface211functions to display processed and/or unprocessed data produced by the system100, settings of the system100, information obtained from tag identifying system180, or any other suitable information. Alternatively, the processor220may not be coupled to a user interface211, and/or can comprise a linking interface230configured to facilitate transfer of processed and/or unprocessed data produced by the system100, settings of the system100, information obtained from a tag identifying system180, or any other appropriate information to a device external to the system100.

The linking interface230is preferably a wired connection, wherein the linking interface230is configured to couple to a wired connector. The linking interface230can facilitate one-way and or two-way communication between system elements and the processor, and can communicate with the processor via inter-integrated circuit communication (I2C), one-wire, master-slave, or any other suitable communication protocol. However, the linking interface230can transmit data in any other way and can include any other type of wired connection (such as a USB wired connection) that supports data transfer between system elements and the processor220. Alternatively, the linking interface230can be a wireless interface. In a wireless variation of the linking interface230, the linking interface230can include a Bluetooth module that interfaces with a second Bluetooth module coupled to another element over Bluetooth communications. The linking interface230of the wireless variation can alternatively implement other types of wireless communications, such as Wi-Fi, 3G, 4G, radio, or other forms of wireless communication.

Other elements of the system100can include a storage module240, which functions to provide local system storage of data. Variations of the system100including a storage module thus allow data to be stored locally prior to transferring the data to an element external to the system. In a specific example, the storage module can provide local storage adequate to accommodate storage of up to10runs of the system100per day, for a month period of time.

1.7 System—Specific Examples

In a first specific example, as shown inFIGS.1B-1D, the platform130is situated intermediately between the first illumination subsystem111comprising a bright-field subsystem and the second illumination subsystem121comprising a fluorescence subsystem, wherein the first light source112of the first illumination subsystem111is configured to transmit light through a first set of optics113directly toward an imaging substrate350, at the platform130, located superior to the first illumination subsystem111. Light from the first light source112and transmitted through the imaging substrate350is then directed toward an optical sensor150at a location superior to the platform130, through a filter module140. Furthermore, the second light source122of the second illumination subsystem121is configured to transmit light toward a mirror102to be reflected at a 90° angle into a second set of optics123through at least one excitation filter141of the filter module140, which reflects by a 90° angle at a dichroic mirror142of the filter module140, through a focusing an optics module160and toward the imaging substrate350located inferior to the second illumination subsystem121, at the platform130. In the first specific example, light from at least one target object at the imaging substrate350is then configured to be transmitted through the focusing and optics module160, directly through the dichroic mirror142, and toward the optical sensor150situated superior to the filter module140. In the first specific example, the filter module140is one of nine filter modules140coupled to a filter stage149defining a substantially circular geometry, with apertures defined within the filter stage149to allow light transmission through the apertures. The filter stage149defines a plane substantially parallel to a plane defined by the platform130, and the filter stage149is located at a position superior to that of the platform130. In the first specific example, each filter module140of the three filter modules can be rotated into alignment with the second light source122of the second illumination subsystem121, thereby allowing light from the second light source122to be transmitted through at least one excitation filter141of a filter module140and to be reflected at a 90° angle by a dichroic mirror142toward a target object at the platform130, and allowing light from the target object to be transmitted through an emission filter143of the filter module140toward the optical sensor150superior to the filter stage149. As such, alignment of a filter module140in the first specific example aligns the excitation filter141with the second light source122, and simultaneously aligns the emission filter143with the optical sensor150.

In the first specific example, the platform130comprises nine guides138arranged in a uniformly distributed circular array, each guide138proximal to a retainer139that holds an imaging substrate350at the platform130. The platform130in the first specific example is further coupled to a platform control module133comprising a translation stage334configured to translate the platform130in coordinate directions parallel to the platform130(e.g., X, Y directions), by way of a translation controller335that automates translation of the translation stage334. The platform control module133in the second specific example further includes an actuator configured to angularly displace the platform130about an axis perpendicular to the platform, thereby rotating one of multiple imaging substrates350with target objects into desired positions for observation and analysis. In variations of the first specific example, the platform control module133can additionally or alternatively be configured to rotate the platform130about an axis parallel the platform to generate a distribution of focal lengths across the platform130for calibration of the relative locations of the optical sensor150and the target object(s) at the platform130, thereby facilitating achievement of a desired focal length to analyze the target object(s). Variations of the first specific example can, however, be configured in any other suitable manner.

In a second specific example, as shown inFIGS.2A and2B, the platform130is situated intermediately between the first illumination subsystem111comprising a bright-field subsystem and the second illumination subsystem121comprising a fluorescence subsystem, wherein the first light source112of the first illumination subsystem111is configured to transmit light through a first set of optics113directly toward an imaging substrate350located inferior to the first illumination subsystem111, at the platform130. Light from the first light source112and transmitted through the imaging substrate350is then directed toward an optical sensor150at a location inferior to the platform130, through a filter module140. Furthermore, the second light source122of the second illumination subsystem121is configured to transmit light through a second set of optics123through at least one excitation filter141of the filter module, which reflects by a 90° angle at a dichroic mirror142of the filter module140, through a focusing an optics module160and toward the imaging substrate350located superior to the second illumination subsystem121, at the platform130. In the second specific example, light from at least one target object at the imaging substrate350is then configured to be transmitted through the focusing and optics module160, directly through the dichroic mirror142, and toward the optical sensor150situated inferior to the filter module140. In the second specific example, the platform130comprises eight guides138arranged in a 2×4 array, each guide138proximal to a retainer139that holds an imaging substrate350at the platform130.

The platform130in the second specific example is further coupled to a platform control module133comprising a translation stage334configured to translate the platform130in coordinate directions parallel to the platform130(e.g., X and Y directions), by way of a translation controller335that automates translation of the translation stage334. The translation stage334and translation controller335of the platform control module133can translate the platform in an X direction by a span of 9″ and in a Y direction by a span of 5″ in the second specific example. The platform control module133in the second specific example further includes an actuator configured to angularly displace the platform130about an axis parallel the platform to generate a distribution of focal lengths across the platform130for calibration of the relative locations of the optical sensor150and the target object(s) at the platform130, thereby facilitating achievement of a desired focal length to analyze the target object(s). Variations of the second specific example can, however, be configured in any other suitable manner.

The system100of the preferred embodiment and variations thereof can be embodied and/or implemented at least in part as a machine configured to receive a computer-readable medium storing computer-readable instructions. The instructions are preferably executed by computer-executable components preferably integrated with the system100and one or more portions of the processor220. The computer-readable medium can be stored on any suitable computer-readable media such as RAMs, ROMs, flash memory, EEPROMs, optical devices (CD or DVD), hard drives, floppy drives, or any suitable device. The computer-executable component is preferably a general or application specific processor, but any suitable dedicated hardware or hardware/firmware combination device can alternatively or additionally execute the instructions.

As a person skilled in the art will recognize from the previous detailed description and from the figures and claims, modifications and changes can be made to the preferred embodiments of the invention without departing from the scope of this invention defined in the following claims.