The assignee for this patent application is
Reporters obtained the following quote from the background information supplied by the inventors: "This invention relates to systems and methods for imaging sample materials within a microfluidic device during an assay reaction process.
"The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying DNA.
"With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of the DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning--A Laboratory Manual (3rd Ed.), Vols. 1-3,
"In some applications, it is important to monitor the accumulation of DNA products as the amplification process progresses. Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the amplification process over time allows one to determine the efficiency of the process, as well as estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide,
"More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
"Further examples of systems, methods, and apparatus for high throughput approaches to performing PCR and other amplification reactions are described in the following publications that are related to the subject matter of the present disclosure.
"U.S. Patent Application Publication No. 2008/0176230 to Owen et al. entitled 'Systems and methods for real-time PCR' (the '230 publication'), the disclosure of which is hereby incorporated by reference, describes systems and methods for the real-time amplification and analysis of a sample of DNA within a micro-channel.
"U.S. Patent No. 7,629,124 to Hasson et al. entitled 'Real-time PCR in micro-channels' (the '124 patent') the disclosure of which is hereby incorporated by reference, describes systems and methods for performing real time PCR in micro-channels by continuously moving boluses of test solution separated by carrier fluid through the micro-channels and performing a process, such as PCR, on each bolus and measuring signals, such as fluorescent signals, at different locations along a defined section of the channel.
"U.S. Patent No. 7,593,560 to Hasson et al. entitled 'Systems and methods for monitoring the amplification and dissociation behavior of DNA molecules' (the '560 patent'), the disclosure of which is hereby incorporated by reference, describes the use of sensors for monitoring reactions within microfluidic channels. The sensor has a defined pixel array for collecting image data, and image data from a select window of pixels (a sub-set of the entire array), which encompasses a portion of interest of a micro-channel, is processed and stored for each of the micro-channels.
"Once there are a sufficient number of copies of the original DNA molecule, the DNA can be characterized. One method of characterizing the DNA is to examine the DNA's dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA) with increasing temperature. The process of causing DNA to transition from dsDNA to ssDNA is sometimes referred to as a 'high-resolution temperature (thermal) melt (HRTm)' process, or simply a 'high-resolution melt' process.
"To monitor a PCR process and/or a melting process (quantitatively and/or qualitatively), an imaging system may be employed to measure an optically detectable characteristic, such as fluorescence, of a dye that is incorporated into the sample material and that varies in a detectable manner as the number of copies of the original DNA molecule increases and/or as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA) with increasing temperature. The accuracy and reliability of nucleic acid assays depends, to a large extent, on the accuracy and precision of such imaging systems. Moreover, the costs of such imaging systems are a significant portion of the cost of an overall instrument for performing nucleic acid assays.
"Thus, there is a continuing need for improvements in accuracy, precision, and cost effectiveness of imaging systems for monitoring nucleic acid diagnostic assays and other biological processes."
In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "Using a 2 dimensional CMOS or CCD sensor to image a fluorescence source is known in biological studies. Usually a microscope lens is used to image the fluorescence source into scientific CMOS/CCD sensor. In accordance with aspects of the present invention, improved imaging and/or lower cost imaging systems are achieved by using a digital single lens reflex ('DSLR') camera as the imaging device in combination with LED excitation sources of a prescribed configuration and arrangement described herein. Such an imaging system has many advantages, including:
"(1) The large CMOS sensor of the DSLR camera allows both flow tracking and thermal melt measurements using the same camera.
"(2) The 8-bit JPEG format of the DSLR camera saves data transfer bandwidth and hard drive spaces compared with 14-bit RAW data. Bit depth can be restored by averaging many pixels.
"(3) A sensor with large pixel density, such of the DSLR camera, permits reactions in the microfluidic channel to be observed. Information such as bubble formation could be obtained to have better control of the PCR and thermal melt process.
"(4) Due to large CMOS sensor size of the DSLR camera, reference fluorescence materials could be used to correct for and remove fluctuations from the light source and the heater.
"Thus, aspects of the invention are embodied in an imaging system configured to generate images of a reaction within a microchannel of a microfluidic device. In one embodiment, the imaging system comprises a sensor element configured to generate a storable image of at least a portion of a microchannel and a plurality of illumination elements disposed with respect to the sensor element and configured to illuminate a portion of the microfluidic chip to be imaged by the sensor element. At least one of the illumination elements comprises an illumination assembly comprising an LED, a mask disposed in front of the LED and having an opening formed therein so as to control an area illuminated by the illumination assembly, a filter along an optic path of the illumination assembly for controlling the spectral content of light emitted by the illumination assembly, and a lens for imaging an area with light emitted by the illumination assembly. The LED, the mask, the filter, and the lens are aligned along an optic axis of the illumination assembly.
"In one embodiment, at least two of the illumination elements are configured to illuminate different portions of the microfluidic chip.
"In another embodiment, the sensor element comprises a digital single lens reflex camera.
"In another embodiment, each of the illumination elements comprises an LED.
"In another embodiment, the imaging system comprises four illumination elements disposed at 90-degree angular increments about the sensor element.
"In another embodiment, the microchannel comprises a first zone and a second zone, wherein a first one of the LEDs is positioned and oriented to illuminate the second zone, a second and a third of the LEDs are spaced 180-degrees from each other and are disposed on opposed sides of the sensor and are positioned and oriented to illuminate the first zone, and wherein a fourth one of the LEDs is positioned and oriented to illuminate both the first zone and the second zone.
"In another embodiment, the sensor element comprises a CMOS sensor.
"In another embodiment, the CMOS sensor has a pixel array of up to 5616.times.3744 pixels or higher.
"In another embodiment, the sensor element includes a pixel array, and the system further comprises logic elements configured to detect an image of only a portion of the pixels of the pixel array.
"In another embodiment, the sensor element is configured to generate multiple images at a frequency of up to 30 Hz or higher.
"In another embodiment, the sensor element is configured to generate an image having an 8-bit JPEG format.
"In another embodiment, the imaging system further comprises reference fluorescence material positioned to be imaged by the sensor element along with at least a portion of a microchannel.
"In another embodiment, the imaging system further comprises at least one extension tube between the sensor and the microchannel. In other embodiments, lens combinations may be used in place of extension tubes.
"In another embodiment, the imaging system further comprises an emission filter positioned between the sensor and the microchannel and is configured to allow light signals of only a selected wavelength to reach the sensor.
"In another embodiment, each of the plurality of illumination elements is configured to illuminate a portion of the microfluidic chip at a prescribed wavelength.
"Further aspects of the invention are embodied in a system for performing a nucleic acid diagnostic assay on a sample material. In one embodiment, the system comprises microfluidic means including micro-channels for transporting sample material and for enabling an assay process to be performed on sample material within one or more portions of the micro-channels. The assay process includes at least one of PCR amplification and/or thermal melt analysis. The system further includes means in operative cooperation with the microfluidic means for introducing sample material into the micro-channels of the microfluidic means; means in operative cooperation with the microfluidic means for moving sample material through each micro-channel of the microfluidic means, thermal means for heating and/or cooling one or more portions of the microfluidic means to one or more selected temperatures, and imaging means for imaging sample material within one or more portions of each micro-channel, including means for storing data related to images created by the imaging means.
"In another embodiment, the system further comprises processing means for processing data related to images created by the imaging means and for generating data relating to results of at least one of the PCR amplification and the thermal melt analysis.
"In another embodiment, the system further comprises control means for controlling operation of the means for introducing sample material, the means for moving sample material, the thermal means, the imaging means, and the processing means.
"In another embodiment, the imaging means is configured for detecting fluorescent emissions of prescribed wavelengths from sample material within the micro-channels and comprises means for directing an excitation signal of a prescribed excitation wavelength at a portion of the micro-channel and means for capturing an image of fluorescent emission of a prescribed emission wavelength from the sample material within the portion of the micro-channel.
"Further aspects of the invention are embodied in a computer-implemented method for analyzing thermal melt data from an image of a reaction within a microchannel of a microfluidic device. In one embodiment, the method comprises the steps of illuminating at least a portion of the microchannel, generating, with a pixel array sensor, an image of fluorescence emitted by material within the illuminated portion of the microchannel, wherein each pixel of the image has a JPEG value, defining a region of interest ('ROI') comprising a portion of the pixels of the pixel array, and calculating the intensity of fluorescence emitted by material within the microchannel by averaging the JPEG values of all the pixels in the ROI.
"In another embodiment, each pixel has two sub-pixels of a first color and one sub-pixel of a second color, and the JPEG value of each pixel is computed as (2.times.first color sub-pixel+1.times.second color sub-pixel)/3.
"In another embodiment, the method further comprises dividing at least a portion of the ROI into one or more sub-ROIs and calculating fluorescence intensity within each sub-ROI by averaging JPEG values of all the pixels in the sub-ROI.
"In another embodiment, the generating step is performed with a CMOS sensor.
"In another embodiment, the CMOS sensor comprises a digital single lens reflex camera.
"In another embodiment, the method further comprises repeating the generating and calculating steps one or more times over a period of time and monitoring changes in the calculated fluorescence intensity over a period of time.
"In another embodiment, the method further comprises monitoring a fluid flow within the microchannel by generating images of fluorescence emitted by material within the illuminated portion of the microchannel at two different times, detecting displacement of a feature of the image within from one image to the next, and computing a time lapse from one image to the next.
"In another embodiment, the method further comprises illuminating a reference fluorescence material positioned adjacent the microchannel, generating, with the pixel array sensor, an image of fluorescence emitted by the reference fluorescence material, and defining at least two ROIs, wherein one ROI encompasses the portion of the microchannel and a second ROI encompasses a portion of the reference fluorescence material.
"In another embodiment, the method further comprises calculating the intensity of fluorescence emitted by the reference fluorescence material by averaging the JPEG values of all the pixels in the ROI encompassing the reference fluorescence material and adjusting the intensity of fluorescence emitted by material within the microchannel based on the intensity of fluorescence emitted by the reference fluorescence material.
"Further aspects of the invention are embodied in a computer-implemented method for analyzing thermal melt data. In one embodiment, the method comprises recording thermal melt image data as a function of time with an imaging system by saving JPEG images generated by the imaging system with time stamps, recording temperature data as a function of time with a temperature control system, and synchronizing the thermal melt data with the temperature data by sending a static record signal to the imaging system to cause the imaging system to record static image data before thermal melt is started, recording the time of the static image data, and synchronizing the temperature data to the time of the static image data.
"In another embodiment, the method comprises sending the static image signal to the imaging system at the same time an initial heater signal is sent to the temperature control system, so that the thermal melt image data and heaters controlled by the temperature control system will have the same start time, and then synchronizing the temperature data and the thermal melt image data based on a relationship of the time of the static image data to the time stamps of the JPEG images of the thermal melt image data and recorded time of the temperature data.
"These and other features, aspects, and advantages of the present invention will become apparent to those skilled in the art after considering the following detailed description, appended claims and accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
"The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements.
"FIG. 1 is a block diagram illustrating a system in which an imaging system incorporating aspects of the invention can be incorporated.
"FIG. 2 is a perspective view of an imaging system including a sensor and LED layout according to an embodiment of the invention.
"FIG. 3 is a side view of an imaging system including a sensor and LED layout according to an embodiment of the invention.
"FIG. 4 is a schematic view of an LED assembly of the imaging system according to an embodiment of the invention."
For more information, see this patent application: Liang, Hongye; Hasson,
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