Patent Grant
9965866
This invention relates to methods and apparatuses for motion tracking and image registration, and more particularly relates to real-time tracking and correlation of particles from images of the particles captured at discrete time intervals.
Given a set of particle images that are captured at discrete time intervals where the elapsed time between images is too large to resolve individual random particle movement, identifying the same particle in each of the images may not be possible. For example, the location of a particular particle in a first image may, in a second image, be vacant or be occupied by a different particle because within the time between when the first image was captured and when the second image was captured the particles in the image may move. As such, it may be difficult to attribute measurements in two separate images to a common particle.
Methods and apparatuses for tracking and correlating particles from images of the particles captured in discrete time intervals allows measurements to be taken from individual particles among a plurality of particles in a plurality of images. In addition to using a first optical detector to capture “classification” images (e.g., images directed to detecting optical signatures, such as fluorescence emission) of the particles, the system may include a second optical detector, herein referred to as a video detector, that captures frames at a rate faster than the rate at which images are captured by the first optical detector. Such a system is able to more closely track the movement of particles, and therefore is capable of more accurately identifying the same particles in a plurality of distinct images taken over a period of time. As a result, the system may improve the accuracy of data acquired from measurements performed on particles through a plurality of images.
For example, consider a fluorescence optical system for the detection of fluorescent particles in a droplet. Due to droplet surface characteristics, the particle movement within the droplet can be volatile. In order to achieve both a multiplex and high limit of detection, multiple “classification” images at different wavelengths may need to be captured. When capturing these images in succession with a single optical detector, several seconds may pass between the time the first image and the last image are captured due to moving parts (e.g., filter wheel, focal position) and/or capture integration time. It can, therefore, become difficult to correlate the positions of the particles in the first classification image with the positions of the particles in a subsequent classification image (e.g., a second, third, fourth, etc. image) due to the movement of the particles between image captures. The methods and apparatuses disclosed herein address this problem by tracking and correlating particles in images taken at discrete time intervals.
A method is disclosed. In one embodiment, the method may include capturing, using an optical detector, a first and a second image of one or more particles and capturing, using a video detector, a plurality of video frames of the one or more particles. Furthermore, in some embodiments, the method may include identifying, using the processor, a first image position of a particle in the first image of the one or more particles. In some embodiments, the method may include correlating, using the processor, the first image position of the particle in the first image to a second image position of the particle in the second image using the plurality of video frames.
In some embodiments, the method may include time stamping each of the images captured using the optical detector. Furthermore, the method may include time stamping each of the plurality of video frames captured using the video detector. The method may include using the time stamps in correlating, using the processor, the first image position of the particle in the first image to a second image position of the particle in the second image.
In some embodiments, the method may include transforming the identified first image position in the first image from a position in an image coordinate system to a position in a video frame coordinate system to identify a first video frame position. In addition, the method may include identifying a first video frame of the plurality of video frames with a time stamp that is closest to the time stamp of the first image. In some embodiments, the method may include identifying a particle with a position in the identified first video frame that is closest to the first video frame position. In some embodiments, the method may include tracking the movement of the particle from the first video frame to a second video frame to locate a second video frame position of the particle in the second video frame. In addition, the method may include inverse transforming the second video frame position in the video frame coordinate system to a position in the image coordinate system to identify a second image position in the second image.
In some embodiments, the method may include capturing, with the video detector, the plurality of video frames at a frame rate determined through a calibration routine. In some embodiments, the method may include storing the plurality of video frames in a storage device.
In some embodiments, the method may include capturing at least a third image of the one or more particles and correlating the first image position of the particle in the first image to a third image position of the particle in the third image using the plurality of video frames.
In some embodiments of the method the video detector has a frame rate that is at least 3 times faster than a frame rate of optical detector. In some embodiments, the video detector has a frame rate that is at least 3 times faster than a frame rate of optical detector.
In some embodiments, the particles may include two fluorophores, and the optical detector may be configured to capture the first image and the second image at different wavelengths corresponding to the two fluorophores. In addition, in some embodiments, the particles may include three fluorophores, and the optical detector may be configured to capture the first image, the second image, and the third image at different wavelengths corresponding to the three fluorophores.
An apparatus is also disclosed. In some embodiments, the apparatus may include an imaging region configured to hold one or more particles. Furthermore, the apparatus may include a light radiating device configured to illuminate the one or more particles in the imaging region. In some embodiments, the apparatus may include an optical detector configured to capture a first and second image of the one or more particles and a video detector configured to capture a plurality of video frames of the one or more particles. In addition, the apparatus may include a processor, coupled to the optical detector and the video detector, and configured to identify a first image position of a particle in the first image of the one or more particles. The processor may also be configured to correlate the first image position of the particle in the first image to a second image position of the particle in the second image using the plurality of video frames.
In some embodiments, the processor may be further configured to time stamp each of the images captured with the optical detector and time stamp each of the plurality of video frames captured with the video detector. In addition, in some embodiments, the processor may be further configured to transform the identified first image position in the first image from a position in an image coordinate system to a position in a video frame coordinate system to identify a first video frame position. Furthermore, the processor may be configured to identify a first video frame of the plurality of video frames with a time stamp that is closest to a time stamp of the first image and to identify a particle with a position in the identified first video frame that is closest to the first video frame position. The processor may be further configured to track the movement of the particle from the first video frame to a second video frame having a time stamp that is closest to a time stamp of the second image to locate a second video frame position of the particle in the second video frame. In addition, the processor may be configured to inverse transform the second video frame position in the second video frame from a position in the video frame coordinate system to a position in the image coordinate system to identify a second image position in the second image.
In some embodiments, the apparatus may include a dichroic mirror to separate light radiating from the light radiating device from light emitted from the particles in the imaging region. In some embodiments, the apparatus may include an optical component to focus the light radiated from the particles.
In some embodiments, the apparatus may include a filter positioned between the imaging region and the optical detector. The filter may be configured to permit light with a first wavelength to pass from the imaging plane, through the filter, and to the optical detector and to reflect or absorb light with a different wavelength than the first wavelength.
In some embodiments, the video detector may be configured to capture video frames at a rate that is at least three times faster than the optical detector captures images. In some embodiments, the video detector may be configured to capture video frames at a rate that is at least ten times faster than the optical detector captures images. In some embodiments, the processor may be configured to determine, through a calibration routine, the rate at which the video detector captures video frames.
In some embodiments, the processor may be configured to synchronize the optical detector and the video detector with one another in time using hardware mechanisms. In some embodiments, the processor may be configured to synchronize the optical detector and the video detector with one another in time using software mechanisms.
The apparatus of claim 10, where the imaging region is an imaging plane.
The apparatus of claim 10, where the imaging region is a three-dimensional volume.
In certain embodiments, the methods and apparatus disclosed herein use no more than one optical detector and no more than one video detector.
The term “coupled” is defined as connected, although not necessarily directly, and not necessarily mechanically.
The terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise.
The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”) and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, a method or device that “comprises,” “has,” “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more elements. Likewise, a step of a method or an element of a device that “comprises,” “has,” “includes” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features. Furthermore, a device or structure that is configured in a certain way is configured in at least that way, but may also be configured in ways that are not listed.
The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Various features and advantageous details are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known starting materials, processing techniques, components, and equipment are omitted so as not to unnecessarily obscure the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the invention, are given by way of illustration only, and not by way of limitation. Various substitutions, modifications, additions, and/or rearrangements within the spirit and/or scope of the underlying inventive concept will become apparent to those having ordinary skill in the art from this disclosure.
In the following description, numerous specific details are included to provide a thorough understanding of disclosed embodiments. One of ordinary skill in the art will recognize, however, that embodiments of the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.
Although certain embodiments are described herein with respect to microspheres or beads, it is to be understood that the systems and methods described herein may also be used with other particles, including microparticles, gold nanoparticles, quantum dots, nanodots, nanoparticles, nanoshells, nanocrystals, droplets, rare-earth metal particles, magnetic particles, latex particles, cells, micro-organisms, vesicles, organelles, organic matter, non-organic matter, or any other discrete substances known in the art. The particles may be formed from a variety of substances including, latex, polystyrene, agarose, silica, glass, or dextran. In certain embodiments, the particles may be droplets formed as an emulsion, including for example, a water-in-oil emulsion or an oil-in-water emulsion. In particular embodiments, a droplet volume may be approximately 1-5 picoliters, 5-10 picoleters; 10-50 picoliters; 50-100 picoleters; 100-500 picoleters; 500-1,000 picoleters; 1,000-5,000 picoliters; 5,000-10,000 picoleters; 10,000-50,000 picoleters; 50,000-100,000 picoliters; 100,000-500,000 picoleters or 500,000-1,000,000 picoleters. In particular embodiments, a droplet volume may be approximately 1-5 femtoliters, 5-10 femtoliters; 10-50 femtoliters; 50-100 femtoliters; 100-500 femtoliters; 500-1,000 femtoliters; 1,000-5,000 femtoliters; 5,000-10,000 femtoliters; 10,000-50,000 femtoliters; 50,000-100,000 femtoliters; 100,000-500,000 femtoliters or 500,000-1,000,000 femtoliters. In certain embodiments, the droplet volume may be approximately 1.0-1.5 microliters; 1.5-2.0 microliters; 2.0-2.5 microliters; or 2.5-3.0 microliters. The particles may have optical properties including color, fluorescence, or chemiluminescence. The particles may serve as vehicles for molecular reactions. Examples of appropriate particles are illustrated and described in U.S. Pat. No.5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No. 6,057,107 to Fulton, U.S. Pat. No. 6,268,222 to Chandler et al., U.S. Pat. No. 6,449,562 to Chandler et al., U.S. Pat. No. 6,514,295 to Chandler et al., U.S. Pat. No. 6,524,793 to Chandler et al., and U.S. Pat. No. 6,528,165 to Chandler, which are incorporated by reference as if fully set forth herein. The systems and methods described herein may be used with any of the particles described in these patents. In addition, particles for use in method and system embodiments described herein may be obtained from manufacturers such as Luminex Corporation of Austin, Texas. The terms “beads” and “microspheres” are used interchangeably herein.
In addition, the types of particles that are compatible with the systems and methods described herein include particles with fluorescent materials attached to, or associated with, the surface of the particles. These types of particles, in which fluorescent dyes or fluorescent particles are incorporated into the particles in order to provide the classification fluorescence (i.e., fluorescence emission measured and used for determining an identity of a particle or the subset to which a particle belongs), are illustrated and described in U.S. Pat. No. 6,268,222 to Chandler et al. U.S. Pat. No. 6,649,414 to Chandler et al., and U.S. Pat. No. 7,718,262 to Chandler et al., which are incorporated by reference as if fully set forth herein. The types of particles that can be used in the methods and systems described herein also include particles having one or more fluorochromes or fluorescent dyes incorporated into the core of the particles.
Particles that can be used in the methods and systems described herein further include particles that will exhibit one or more fluorescent signals upon exposure to one or more appropriate light sources. Furthermore, particles may be manufactured such that upon excitation the particles exhibit multiple fluorescent signals, each of which may be used separately or in combination to identify the particles.
The methods described herein generally include analyzing one or more images of particles and processing data measured from the images to determine one or more characteristics of the particles such as, but not limited to, numerical values representing the magnitude of fluorescence emission of the particles at multiple detection wavelengths. Subsequent processing of the one or more characteristics of the particles, such as using one or more of the numerical values to determine a token ID representing the multiplex subset to which the particles belong and/or a reporter value representing a presence and/or a quantity of analyte bound to the surface of the particles, can be performed according to the methods described in U.S. Pat. No. 5,736,330 to Fulton, U.S. Pat. No. 5,981,180 to Chandler et al., U.S. Pat. No. 6,449,562 to Chandler et al., U.S. Pat. No. 6,524,793 to Chandler et al., U.S. Pat. No. 6,592,822 to Chandler, U.S. Pat. No. 6,939,720 to Chandler et al., U.S. Pat. No. 8,031,918 to Roth, which are incorporated by reference as if fully set forth herein. In one embodiment, the methods described herein can be used in a MagPix® instrument. The MagPix® instrument is a multiplexing platform with automated image processing software capable of measuring fluorescent intensity of up to 50 optically-distinct populations of magnetic beads randomly distributed in an imaging field.
Turning now to the figures,
According to an embodiment, the particle imaging device 300 may include an imaging plane 302 configured to hold a plurality of particles, and a light radiating device 304 to illuminate the particles on the imaging plane 302. In one embodiment, the light radiating device 304 may be an infrared illuminator. In some embodiments, the light radiating device may be an LED, laser, or multispectral lamp. A dichroic mirror 312 may be used to separate the light radiating from the light radiating device 304 from the light emitted, and thus radiated, from the particles on the imaging plane 302 after being illuminated by the light radiating device 304.
The particle imaging device 300 may also include an optical detector 310 configured to capture a plurality of images of the particles on the imaging plane 302, and a video detector 306 configured to capture a plurality of video frames of the particles. The light radiating from the particles on the imaging plane 302 may be focused by an optical component 316, split by beam splitter 314, and directed to the optical detector 310 and the video detector 306. In order to produce more reliable images, for example, the particles may be at least partially immobilized on the imaging plane 302 prior to capturing an image with the optical detector 310 or a video frame with the video detector 306 of the particles.
The particle imaging device 300 may further include a filter 308 disposed between the imaging plane 302 and the optical detector 310 to filter the plurality of images prior to being captured by the optical detector 310. In some embodiments, the filter 308 may be configured to permit light with a first wavelength to pass from the imaging plane 302, through the filter 308, and to the optical detector 310, and to reflect or absorb light with a different wavelength than the first wavelength. This allows measurements to be performed on particles based on their fluorescence at a particular wavelength. In one embodiment, each image of the particles captured by the optical detector 310 may be an image of the particles at a particular wavelength. For example, a first image of the particles may show only particles that radiate light at a first wavelength, and a second image of the particles may show only particles that radiate light at a second wavelength. In some embodiments, a particle may radiate light at multiple wavelengths, and therefore may appear in multiple images of particles filtered at different wavelengths, such as the first and second images. Because one use of the video detector 306 may be to track and resolve the position of particles, the video detector may capture video frames of particles at more than one wavelength. As such, particles may be visible to the video detector even if they are not visible to the optical detector due to filtering. Therefore, a particle's position may be tracked using the video detector even if the particle does not emit light at a wavelength that reaches the optical detector.
The particle imaging device 300 may further include a processor 318 that is coupled to the optical detector 310 and the video detector 306, and that is configured to synchronize the optical detector 310 with the video detector 306. The processor 318 may synchronize, in time, the optical detector 310 with the video detector 306 by time stamping each of the plurality of images captured with the optical detector 310, and time stamping each of the plurality of video frames captured with a video detector 306. The processor 318 may also be configured to synchronize the optical detector 310 and the video detector 306 with one another in time using hardware, such as an electrical connection that carries a synchronizing pulse. In some embodiments, the video detector 306 may be the same type of detector as the image detector. However, in some embodiments, the video detector 306 may have less resolution than the image detector 310. Furthermore, the video detector 306 may be configured to capture video frames at a rate faster than the optical detector 310 captures images. By capturing video frames at a rate faster than images are captured by the optical detector 310, the video detector 306 may allow the processor to more closely track the movement of particles between the time instants when images are captured with the optical detector 310. For example, the video detector 306 may start capturing video frames when the first image is captured by the optical detector 310, and may continue capturing video frames at a faster rate than the optical detector 310 captures images until the optical detector 310 captures its last image of the particles. Because the video frames have less time between successive frames as compared to images taken with the optical detector, the movement of particles may be tracked using the relative locations of the measured particles. The rate at which the video frames are captured may be determined through numerous methods. For example, in one embodiment, the processor 318 may be configured to determine the rate at which the video detector 306 captures video frames through a calibration routine. Such a calibration routine may, for example, acquire video frames at different frame rates and determine a minimum frame rate that allows for accurate tracking of particles given the amount and type of movement of the particles. The frame rate may then be increased by a predetermined amount to ensure that particles are accurately tracked. In another embodiment, the processor 318 may be configured to determine the rate at which the video detector 306 captures video frames based on known design characteristics, in which case the rate is predetermined to be sufficiently fast to accurately capture particle movement. For example, in some embodiments, the video frame rate may be three times the rate at which images are captured, which would allow for two video frames between two image captures. In some embodiments, the video frame rate may be ten times as fast as the frame rate of the image detector, which would provide nine video frames between two image captures. Larger video frame rates may allow for better tracking of particles, but may also require more processing and storage resources. The particle imaging device 300 may also include a storage device 320 to store the plurality of video frames captured by the video detector 306 and/or the images captured by the optical detector 310.
The processor 318 may also be configured to identify a first image position Ip1 of a particle X in a first image I1 captured by the optical detector 310, and to correlate the first image position Ip1 of the particle X to a second image position Ip2 of the particle X in a second image I2 captured by the optical detector 310 using the plurality of video frames. Locations in an image captured by an optical detector 310 may be specified in an image coordinate system and locations in a video frame captured by a video detector 306 may be specified in a video frame coordinate system. For example, if the video detector 306 has less resolution than the optical detector 310, different coordinate systems may be used to describe locations (or particle positions) in images taken by the two detectors. A particle may show up in both images, but may have different coordinates in the two images because the two images have different resolution, and therefore different coordinate systems. The processor 318 may be configured to transform the identified first image position Ip1 in the first image I1 from a position in the image coordinate system to a position in the video frame coordinate system. By transforming the location coordinates from the image coordinate system to the video frame coordinate system, a first video frame position Fp1 may be identified, wherein the first video frame position Fp1 is the position in a video frame that corresponds to the first image position Ip1 in an image.
In order to correlate particles from separate images captured by the optical detector 310, the relevant video frames may be analyzed. For example, the relevant video frames may be the video frames that are captured approximately between the time that the first image Ii is captured by the optical detector 310 and the time that the second image I2 is captured. The relationship between the first image I1 and the second image I2 captured by the optical detector 310 may not be critical because the first and second images, I1 and I2 respectively, only distinguish that the first image I1 and the second image I2 are two separate images captured by the optical detector 310. For example, the second image I2 may be the fourth image captured by the optical detector 310 after the first image I1 was captured. As another example, the second image I2 may be captured before the first image I1 is captured, which may be the case if data is processed after the images are taken and the processor uses an image taken later in time as a starting point. Nevertheless, the most relevant video frame to the first image I1 may be the video frame that is captured within the shortest amount of time either before or after the first image I1 was captured. Therefore, the processor 318 may be further configured to identify a first video frame F1, of the plurality of video frames captured by the video detector 306, with a time stamp that is closest to the time stamp of the first image I1 captured by the optical detector 310.
In order to track the movement of a particular particle X using the video frames, the particle X may be identified in the first video frame F1. Therefore, the processor 318 may also be configured to identify a particle Y with a position in the identified first video frame F1 that is closest to the first video frame position Fp1. A particle Y in the first video frame F1 may be the same particle as particle X in the first image I1, therefore particle X and particle Y will hereinafter be referred to as particle X. To track the movement of particle X from the time when the first image I1 was captured to the time when the second image 12 was captured, the processor 318 may be configured to track the movement of particle X from the first video frame F1 to a second video frame F2 to locate a second video frame position Fp2 of particle X in the second video frame F2. In an embodiment, the second video frame F2 may be a video frame with a time stamp that is closest to the time stamp of the second image 12 captured by the optical detector 310, even if addition video frames were taken between the first video frame F1 and the second video frame F2. Tracking the movement of particle X may, in some embodiments, include tracking particle X in successive video frames either in real time or in software post-processing step performed by the processor.
With the second video frame position Fp2 of the particle X identified, the processor 318 may be configured to inverse transform the second video frame position Fp2 in the second video frame F2 from a position in the video frame coordinate system to a position in the image coordinate system to identify a second image position Ip2 of particle X in the second image I2. By transforming the location coordinates from the video frame coordinate system back to the image coordinate system, the second image position Ip2 of particle X in the second image I2 may be identified as the position of the particle in the second image I2 that is closest to the second image position Ip2 for particle X that was identified from the inverse transform operation.
To track the movement of particle 611 as it moves from its location 611A in the first image 602 to its location 611B in the second image 604, at block 406, a plurality of video frames of the one or more particles may be captured with the video detector. For example,
To identify a particle of interest that may be tracked using a plurality of video frames, a first image position of a first particle in the first image 602 of the one or more particles may, at block 408, be identified. For example, the first particle may be particle 611 from the first image 602, and its first image position may be 611A. With the particle of interest identified as particle 611, the first image position 611A of a first particle 611 in the first image 602 may, at block 410, be correlated to a second image position of the first particle 611 in the second image using the plurality of video frames. For example, the second image may be the second image 604 of
At block 504, a first video frame with the time stamp that is closest to the time stamp of the first image 602 may be identified. For example, the video frame that captures particle 611 at position 611C may be identified as the first video frame with the time stamp that is closest to the time stamp of the first image 602. At block 506, a particle with a video frame position in the identified first video frame that is closest to the first video frame position may be identified. For example, the particle identified at block 506 may be particle 611 because the video frame position 611C of particle 611, which was captured by the first video frame identified at block 504, is closest to the first video frame position identified at block 502. Therefore, because the particle identified at block 506 may, in some embodiments, also be the same particle as particle 611, except in the video frame coordinate system as opposed to in the image coordinate system, the particle identified at block 506 will hereinafter be referred to as particle 611 as well. This is a desired result because the particle 611 being tracked with the plurality of video frames should be the same as the particle 611 identified in the first image 602 so that the particle's second image position 611B in a second image 604 may be correlated with the first image position 611A of the particle 611 in the first image 602.
The movement of particle 611 may, at block 508, be tracked from the first video frame to a second video frame to locate a second video frame position of the particle 611 in the second video frame. In some embodiments, particles may be tracked by using absolute locations in a sequence of images and assuming that each particle in an image that is closest to the position of a particle in previous image corresponds to the same particle. If the sample rate of the video image is fast enough, such assumption should be true. Referring to
For the second image captured by the optical detector, two video frames with two time stamps that are closest to the time stamp of the second image may, at block 908, be identified. For each of the two video frames identified at block 908, the video frame position of a particle that is closest to the first video frame position in each of the two video frames may, at block 910, be identified. The video frame position of the two particles in each of the two video frames captured at block 908 may, at block 912, be interpolated to determine the first particle's second video frame position in the video frame coordinate system. At block 914, an inverse transform may be applied to the second video frame position to determine the first particle's image position in the second image. The first particle's image position in the second image may be established as the position of a particle in the second image that is closest to the image position that was a result of the inverse transform operation at block 914. However, because images taken by the image detector may be filtered (only certain wavelengths allowed to reach the detector) the first particle may not show up in the second image. However, because the location in the second image is known, the fact that the particle does not emit light that is being captured in the second image can be known and that information can be attributed to the first particle. In some embodiments, if no particle is found within a predetermined radius of an expected location of a particle in an image, it may be determined that that particle does not emit light in the wavelength being detected. By determining the first particle's second image position and the first particle's first image position, information about that particle taken in the first and second images may be correlated (even if the particle does not show up in one of the images).
The methods and apparatus described herein are generally described as being implemented with particles in an imaging plane. However, the methods and apparatus may also be used with particles in other imaging regions. For example, imaging regions may include three dimensions regions or volumes, such as a droplet or a microwell. In addition, multiple detectors may be used with the methods described herein. For example, two or more video detectors placed at different locations may be used to track the positions of particles moving in three dimensions. That tracking information may then be used to associate measurements taken by one or more optical detectors, at different times, to a particular particle.
In some embodiments, tangible computer-readable media, such as CDs, hard-disks, RAM, or Flash memory, for example, may be made, recorded or written to with instructions, that when executed by a processor, are capable of performing the methods described herein.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the apparatus and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. For example, the methods disclosed herein may be applied in real time as information is being gathered, or it can be performed after all measurements have been taken in a post-processing step. In addition, modifications may be made to the disclosed apparatus, and components may be eliminated or substituted for the components described herein where the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present processes, disclosure, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
This application is a continuation application of U.S. application Ser. No. 14/200,322, filed Mar. 7, 2014, which claims priority to U.S. Provisional Patent Application Ser. No. 61/790,327, filed Mar. 15, 2013, the contents of which are incorporated by reference herein.
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