EP-4345444-B1 - DYNAMIC OPTICAL SYSTEM CALIBRATION

EP4345444B1EP 4345444 B1EP4345444 B1EP 4345444B1EP-4345444-B1

Inventors

BLAIR, DUSTIN
BARTIG, Kevin
SIM, Daeyong
WEN, Patrick
EARNEY, John
PRABHU, Anmiv
ABASKHARON, Rachel
HOLST, Gregory
LIU, Chia-Hsi
THAKUR, Ravi Bhushan Singhchawhan
WATSON, Dakota

Dates

Publication Date: 20260506
Application Date: 20230928

Claims (13)

An apparatus, comprising: a flow cell (300) comprising one or more channels (310), wherein each of the one or more channels has a length and a width with the length being greater than the width, and comprises a surface having a plurality of reaction sites; an imaging assembly (122) to receive light emitted from a reactant positioned at the reaction sites in response to an excitation light; a focus component (162) to, for each channel from the one or more channels, obtain image quality proxy values for the surface of that channel; and logic circuitry comprising a first memory, wherein the logic circuitry is configured to, for a subject channel from the one or more channels: for each of a subject plurality of regions of interest, wherein each region of interest is a two dimensional region on the surface of the subject channel having a plurality of reaction sites separated from each other along the length of the subject channel and a plurality of reaction sites separated from each other along the width of the channel, perform a set of calibration acts comprising: capturing, using the imaging assembly, an image of that region of interest; storing the image of that region of interest in a first memory; determining one or more image quality proxy values for that region of interest using the focus component; and calculating an image quality score for that region of interest; generate a calibration curve relating image quality scores for the regions of interest to image quality proxy values for the regions of interest; and while driving relative motion of the subject channel and a field of view of the imaging assembly along the length of the subject channel via one or more actuators of a sample stage on which the flow cell is mounted or via one or more actuators of the imaging assembly, perform a set of base calling acts comprising: obtaining nucleotide data based on using the imaging assembly to detect light emitted from reactants positioned at reaction sites on the surface of the subject channel; obtaining one or more image quality proxy values using the focus component while obtaining nucleotide data; and based on the calibration curve and on the one or more image quality proxy values obtained while obtaining nucleotide data, determining whether to adjust a feature of the imaging assembly wherein: the logic circuitry comprises: a programmed general purpose processor; and processor-less special purpose logic circuit; the first memory is a local memory resident on the processor-less special purpose logic circuit; the apparatus comprises a second memory operatively connected to the programmed general purpose processor; for each region of interest from the subject plurality of regions of interest the set of calibration acts comprises, before storing the image of that region of interest in the first memory, store the image of that region of interest in the second memory; for an initial region of interest from the subject plurality of regions of interest, storing the image of that region of interest in the first memory comprises transferring the image of that region of interest from the second memory to the first memory; for each region of interest from the subject plurality of regions of interest other than the initial region of interest, storing that region of interest in the first memory comprises: transferring a first portion of the image of that region of interest from the second memory to the first memory at a time when the first memory already contains a second portion of that region of interest as a result of that second portion being comprised by a different, previously stored, region of interest, wherein the first portion of the image and that region of interest and the second portion of that region of interest combine to provide the image of the region of interest; and removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; the processor-less special purpose logic circuit is configured to, for each region of interest from the plurality of regions of interest, calculate the image quality score for that region of interest.
The apparatus of claim 1, wherein for each region of interest from the subject plurality of regions of interest, that region of interest overlaps with at least one other region of interest from the subject plurality of regions of interest along the length of the subject channel.
The apparatus of claim 1, wherein, for at least one region of interest from the subject plurality of regions of interest, at least a portion of the second portion that region of interest is comprised by a plurality of different, previously stored, regions of interest.
The apparatus of claim 1, wherein, for each of the subject plurality of regions of interest: capturing, using the imaging assembly (122), the image of that region of interest comprises capturing a corresponding image of the subject channel, wherein: the corresponding image of the subject channel has an extent along the width of the subject channel greater than an extent of the region of interest along the width of the subject channel; and the corresponding image of the subject channel has an extent along the length of the subject channel equal to an extent of the region of interest along the length of the subject channel; and storing the image of that region of interest in the second memory comprises storing the corresponding image of the subject channel in the second memory.
The apparatus of claim 1, wherein: each of the one or more channels (210, 310) comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region; the logic circuitry is configured to: during a first period, move a field of view of the imaging assembly along the length of the subject channel from the first end region of the subject channel, through the intermediate region of the subject channel, to the second end region of the subject channel; perform the set of calibration acts with a first plurality of regions of interest as the subject plurality of regions of interest during the first period, wherein the first plurality of regions of interest are regions of interest in the first end region of the subject channel; during a second period, move a field of view of the imaging assembly along the length of the subject channel from the second end region of the subject channel, through the intermediate regions of the subject channel, to the first end region of the subject channel; and perform the set of calibration acts with a second plurality of regions as the subject plurality of regions of interest during the second period, wherein the second plurality of regions of interest are regions of interest in the second end region of the subject channel.
The apparatus of claim 5, wherein the logic circuitry is configured to perform the set of calibration acts with a third plurality of regions of interest as the subject plurality of regions of interest, wherein the third plurality of regions of interest are regions of interest in the intermediate region of the subject channel.
The apparatus of claim 6, wherein the logic circuitry is configured to: while performing the set of calibration acts with the first plurality of regions of interest as the subject plurality of regions of interest, drive relative movement between the feature of the imaging assembly (122) and the flow cell (110) along a height, wherein the height is perpendicular to the length and width of the subject channel, through a continuous range of motion between a first value and a second value; and while performing the set of calibration acts with the third plurality of regions of interest as the subject plurality of regions of interest, drive relative movement between the feature of the imaging assembly (122) and the flow cell (110) along the height through a continuous range of motion between a third value and a fourth value, wherein the third value and the fourth value are each between the first value and the second value.
The apparatus of claim 1, wherein: the feature of the imaging assembly (122) is an objective lens (142); the logic circuitry is configured to, for the subject channel from the one or more channels (210, 310), while performing the set of calibration acts: drive relative movement between the objective lens (142) and the surface of the subject channel through a continuous range of motion along a height which is perpendicular to the length and the width of the subject channel; and drive relative motion of the subject channel and the field of view of the imaging assembly (122) along the length of the subject channel; the focus component (162) is configured to, for each channel from the one or more channels, obtain image quality proxy values for the surface of that channel by performing acts comprising projecting a set of spots onto the surface of the channel, and detecting reflections of the set of spots from the surface of that channel; for each region of interest from the subject plurality of regions of interest: the one or more image quality proxy values for that region of interest comprise an average spot separation value for that region of interest; and determining the one or more image quality proxy values for that region of interest using the focus component comprises the focus component projecting the set of spots onto, detecting reflections of the set of spots from, the surface of the subject channel while capturing the image of that region of interest; and determining whether to adjust the feature of the imaging assembly comprises determining whether to adjust relative positions of the objective lens and the surface of the subject channel along the height.
A method comprising: for each of a subject plurality of regions of interest, performing a set of calibration acts, wherein each region of interest is a two dimensional region on a surface of a subject channel of a flow cell (110, 200, 300), the subject channel having a plurality of reaction sites separated from each other along the length of the subject channel and a plurality of reaction sites separated from each other along the width of the channel, perform a set of calibration acts comprising: capturing, using an imaging assembly (122), an image of that region of interest; storing the image of that region of interest in a first memory; determining one or more image quality proxy values for that region of interest using a focus component (162) of a system for analyzing chemical or biological materials; calculating an image quality score for that region of interest; generating a calibration curve relating image quality scores for the regions of interest to image quality proxy values for the regions of interest; while driving relative motion of the subject channel and a field of view of the imaging assembly along the length of the subject channel via one or more actuators of a sample stage on which the flow cell is mounted or via one or more actuators of the imaging assembly, perform a set of base calling acts comprising: obtaining nucleotide data based on using the imaging assembly to detect light emitted from reactants positioned at reaction sites on the surface of the subject channel; obtaining one or more image quality proxy values using the focus component while obtaining nucleotide data; and based on the calibration curve and on the one or more image quality proxy values obtained while obtaining nucleotide data, determining whether to adjust a feature of the imaging assembly; wherein: the first memory is a local memory resident on a processor-less special purpose logic circuit; for each region of interest from the subject plurality of regions of interest the set of calibration acts comprises, before storing the image of that region of interest in the first memory, store the image of that region of interest in a second memory, wherein the second memory is operatively connected to a general purpose processor; for an initial region of interest from the subject plurality of regions of interest, storing the image of that region of interest in the first memory comprises transferring the image of that region of interest from the second memory to the first memory; for each region of interest from the subject plurality of regions of interest other than the initial region of interest, storing that region of interest in the first memory comprises: transferring a first portion of the image of that region of interest from the second memory to the first memory at a time when the first memory already contains a second portion of that region of interest as a result of that second portion being comprised by a different, previously stored, region of interest, wherein the first portion of the image and that region of interest and the second portion of that region of interest combine to provide the image of the region of interest; and removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; the processor-less special purpose logic circuit is to, for each region of interest from the plurality of regions of interest, calculate the image quality score for that region of interest; for at least one region of interest from the subject plurality of regions of interest, at least a portion of the second portion that region of interest is comprised by a plurality of different, previously stored, regions of interest.
The method of claim 9, wherein, for each of the subject plurality of regions of interest: capturing, using the imaging assembly (122), the image of that region of interest comprises capturing a corresponding image of the subject channel, wherein: the corresponding image of the subject channel has an extent along the width of the subject channel greater than an extent of the region of interest along the width of the subject channel; and the corresponding image of the subject channel has an extent along the length of the subject channel equal to an extent of the region of interest along the length of the subject channel; and storing the image of that region of interest in the second memory comprises storing the corresponding image of the subject channel in the second memory.
The method of claim 9, wherein: each of the one or more channels (210, 310) comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region; the method comprises: during a first period, moving a field of view of the imaging assembly along the length of the subject channel from the first end region of the subject channel, through the intermediate region of the subject channel, to the second end region of the subject channel; performing the set of calibration acts with a first plurality of regions of interest as the subject plurality of regions of interest during the first period, wherein the first plurality of regions of interest are regions of interest in the first end region of the subject channel; during a second period, moving a field of view of the imaging assembly along the length of the subject channel from the second end region of the subject channel, through the intermediate regions of the subject channel, to the first end region of the subject channel; and performing the set of calibration acts with a second plurality of regions as the subject plurality of regions of interest during the second period, wherein the second plurality of regions of interest are regions of interest in the second end region of the subject channel; performing the set of calibration acts with a third plurality of regions of interest as the subject plurality of regions of interest, wherein the third plurality of regions of interest are regions of interest in the intermediate region of the subject channel; and determining a nucleotide sequence for a sample of biological material by performing sequencing by synthesis based on nucleotide data captured from the third plurality of regions of interest.
The method of claim 11, wherein the method comprises: while performing the set of calibration acts with the first plurality of regions of interest as the subject plurality of regions of interest, driving relative movement between the feature of the imaging assembly (122) and the flow cell (110, 200, 300) along a height, wherein the height is perpendicular to the length and width of the subject channel, through a continuous range of motion between a first value and a second value; and while performing the set of calibration acts with the third plurality of regions of interest as the subject plurality of regions of interest, driving relative movement between the feature of the imaging assembly and the flow cell along the height through a continuous range of motion between a third value and a fourth value, wherein the third value and the fourth value are each between the first value and the second value.
The method of claim 9, wherein: the feature of the imaging assembly (122) is an objective lens (142); the method comprises for the subject channel, while performing the set of calibration acts: driving relative movement between the objective lens and the surface of the subject channel through a continuous range of motion along a height which is perpendicular to the length and the width of the subject channel; and driving relative motion of the subject channel and the field of view of the imaging assembly along the length of the subject channel; the focus component is to, for the subject channel, obtain image quality proxy values for the surface of that channel by performing acts comprising projecting a set of spots onto the surface of the channel, and detecting reflections of the set of spots from the surface of that channel; for each region of interest from the subject plurality of regions of interest: the one or more image quality proxy values for that region of interest comprise an average spot separation value for that region of interest; and determining the one or more image quality proxy values for that region of interest using the focus component comprises the focus component projecting the set of spots onto, detecting reflections of the set of spots from, the surface of the subject channel while capturing the image of that region of interest; and determining whether to adjust the feature of the imaging assembly comprises determining whether to adjust relative positions of the objective lens and the surface of the subject channel along the height.

Description

BACKGROUND The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves may also correspond to implementations of the claimed technology. Aspects of the present disclosure relate generally to biological or chemical analysis and more particularly to systems and methods using image sensors for biological or chemical analysis. Various protocols in biological or chemical research involve performing a large number of controlled reactions on local support surfaces or within predefined reaction chambers. The designated reactions may then be observed or detected, and subsequent analysis may help identify or reveal properties of chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g., fluorescent label) may be exposed to thousands of known probes under controlled conditions. Each known probe may be deposited into a corresponding well of a flow cell channel. Observing any chemical reactions that occur between the known probes and the unknown analyte within the wells may help identify or reveal properties of the analyte. Other examples of such protocols include known DNA sequencing processes, such as sequencing-by-synthesis (SBS) or cyclic-array sequencing. In some conventional fluorescent-detection protocols, an optical system is used to direct an excitation light onto fluorescently-labeled analytes and to also detect the fluorescent signals that may be emitted from the analytes. Such optical systems may include an arrangement of lenses, filters, and light sources. It may be desirable to provide calibration of such optical systems without substantially affecting overall processing times. US2010/111768 A1 describes systems and devices for sequencing of nucleic acid, such as short DNA sequences from clonally amplified single-molecule arrays. US2010/157086 A1 describes a method and system for controlling focus dynamically of a sample imager, the method comprising scanning a sample with an optical assembly that apportions the sample into regions based on a scan pattern. EP3988985 A2 describes methods and systems for automatically focusing multiple images of one or more objects on a substrate. US2010/208961 A1 describes a method for determining the quality of focus of a digital image of a biological specimen including obtaining a digital image of a specimen using a specimen imaging apparatus. A measure of image texture is calculated at two different scales, and the measurements are compared to determine how much high-resolution data the image contains compared to low-resolution data. US2022/150394 A1 describes a method used to generate an analysis image of a moving sample based on one or more exposures. US2018/258468 A1 describes a laser line illuminator for high throughput sequencing. Imaging systems including an objective lens and a line generation module are described. The objective lens may focus a first light beam emitted by the line generation module and a second light beam emitted by the line generation module at a focal point external to a sample so as to adjust line width. EP3373400 A1 describe systems and methods for improved focus tracking using a hybrid mode light source, the systems and methods including an imaging system that may include a laser diode source; an objective lens positioned to direct a focus tracking beam from the light source onto a location in a sample container and to receive the focus tracking beam reflected from the sample; and an image sensor that may include a plurality of pixel locations to receive focus tracking beam that is reflected off of the location in the sample container, where the reflected focus tracking beam may create a spot on the image sensor. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a schematic diagram of an example of an imaging assembly that may be implemented in a system for biological or chemical analysis.FIG. 2 depicts a perspective view of an example of a flow cell that may be utilized with the system of FIG. 1.FIG. 3 depicts an enlarged perspective view of a channel of the flow cell of FIG. 3.FIG. 4 depicts a top plan view of another example of a flow cell that may be utilized with the system of FIG. 1FIG. 5 depicts an enlarged top plan view of a channel of the flow cell of FIG. 4.FIG. 6 depicts a graph depicting an example of image capture positions during a focus model generation process.FIG. 7 depicts a motion profile depicting an example of an integrated through focus path for moving an objective lens of an imaging assembly for focus model generation and/or updating.FIG. 8 depicts a graph detecting focus tracking spots reflected b