HK1243185B - Optical scanning systems for in situ genetic analysis - Google Patents

Description

Optical scanning system for in situ genetic analysis

Background Art

In conventional (i.e., widefield) fluorescence microscopy, the entire sample is uniformly immersed in light from the light source. All parts of the sample in the light path are excited simultaneously, and the resulting fluorescence is detected by the microscope's photodetectors or a camera that includes a large unfocused background. In contrast, confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signals. Since only light generated by fluorescence very close to the focal plane can be detected, the optical resolution of the image, especially in the direction of sample depth, is much better than that of widefield microscopy. However, since most of the light from the sample fluorescence is blocked at the pinhole, this increased resolution comes at the expense of reduced signal intensity - therefore, long exposure times are usually required.

A disadvantage of some photoluminescence-based scanning instruments (or imaging systems) used in current fluorescence-based synthesis sequencing (SBS) systems is that they have poor confocality (i.e., semi-confocal at best). These semi-confocal systems have a low signal-to-noise ratio (S/N ratio) and are therefore insufficient to eliminate out-of-focus features in the sample. In addition, current dithered focus tracking methods cannot maintain focus during imaging. Therefore, new methods are needed for imaging (or scanning) in photoluminescence-based SBS systems.

Summary of the Invention

Among the various implementations of systems, methods, and apparatus within the scope of the appended claims (each of which has several aspects), no single one is responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein.

Disclosed are systems and methods for performing fluorescence in situ sequencing. Specifically, one embodiment provided herein is a confocal time-delay integration (TDI) line scan imaging system with a high signal-to-noise ratio and high confocality for producing high-resolution images of a sample. In one example, the confocal TDI line scan imaging system includes various pinhole and/or slit aperture mechanisms in front of an image sensor, wherein the various pinhole and/or slit aperture mechanisms are configured to reject out-of-focus light. In another example, the confocal TDI line scan imaging system includes various pinhole and/or slit aperture mechanisms in an intermediate image plane conjugated to the image sensor.

Also provided herein are structures that include focus tracking features that can be used to maintain focus during imaging. In one example, various configurations of focusing strips on a substrate in contact with a tissue sample to be imaged are provided. In another example, the strips are cut into the tissue sample, thereby providing exposed strips that can serve as a substrate for the focus tracking features.

Also provided herein are flow cells and methods for processing tissue samples in a flow cell. That is, various configurations and methods are provided herein in which a tissue sample is placed within a reaction chamber of a flow cell during assembly of the flow cell and then chemical manipulations are performed on the tissue sample.

Also provided herein is a flow cell for performing chemical operations on tissue samples using an open container. In one example, a substantially "dry" imaging process can be used. In another example, a liquid immersion imaging process can be used.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 illustrates a side view of an example of a confocal imaging system according to one embodiment.

FIG. 2 illustrates another configuration of the confocal imaging system shown in FIG. 1 .

3 illustrates a side view of an example of a sensor aperture mechanism of the confocal imaging system shown in FIG. 1 and FIG. 2 .

4A and 4B illustrate side views of another example of a sensor aperture mechanism of the confocal imaging system shown in FIG. 1 and FIG. 2 .

5A and 5B illustrate side views of yet another example of a sensor aperture mechanism of the confocal imaging system shown in FIG. 1 and FIG. 2 .

6A and 6B illustrate a plan view and a cross-sectional view, respectively, of an example of a structure including focus stripes for improved focus tracking during imaging.

7 illustrates a side view of the structure shown in FIGs. 6A and 6B when used in an imaging process.

8 illustrates a side view of another example of a structure including focus strips for improved focus tracking during imaging.

9 illustrates a side view of another technique for providing improved focus tracking during imaging.

10A and 10B illustrate plan and cross-sectional views, respectively, of an example of a flow cell for holding and processing tissue samples.

11 illustrates a flow chart of an example of a method for processing a tissue sample using the flow cell shown in FIG. 10A and FIG. 10B .

12A and 12B illustrate a plan view and a cross-sectional view, respectively, of another example of a flow cell for holding and processing tissue samples.

13A and 13B illustrate additional side views of the flow cell shown in FIG. 12A and 12B , and show tissue samples at different locations within the sequencing chamber.

14A and 14B illustrate a plan view and a cross-sectional view, respectively, of an example of a bonding portion of the flow cell shown in FIG. 12A and FIG. 12B .

15 illustrates a flow chart of an example of a method for processing a tissue sample using the flow cell shown in FIG. 12A and FIG. 12B .

16A and 16B illustrate side views of an example of a flow cell using an open container to hold a tissue sample and an example of a process for imaging the tissue sample "dry" therein.

17A and 17B illustrate side views of the flow cell shown in FIGs. 16A and 16B and the liquid immersion process for imaging a tissue sample therein.

The various features shown in the figures may not be drawn to scale. Accordingly, for clarity, the sizes of the various features may be arbitrarily expanded or reduced. In addition, some drawings may not depict all the components of a given system, method or device.

DETAILED DESCRIPTION

The detailed description set forth below in conjunction with the accompanying drawings is intended as a description of exemplary embodiments of the present invention and is not intended to represent the only embodiments in which the present invention may be practiced. The term "exemplary" as used throughout this specification means "serving as an example, instance, or illustration" and should not be construed as preferred or advantageous over other exemplary embodiments. The detailed description includes specific details to provide a thorough understanding of the exemplary embodiments of the present invention. In some cases, some devices are shown in block diagram form.

Sequencing

The systems and methods described herein can be used in conjunction with various nucleic acid sequencing technologies. These sequencing technologies include, but are not limited to, in situ sequencing technologies for reading sequence information from nucleic acids directly from cells or tissues (Lee, Je Hyuk, et al., "Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues" Nature protocols 10.3 (2015): 442-458; Lee, Je Hyuk, et al., "Highly multiplexed subcellular RNA sequencing in situ" Science 343.6177 (2014): 1360-1363; and Mitra, Robi D, et al., "Fluorescent in situ sequencing on polymerase colonies" Analytical biochemistry 320.1 (2003): 55-65, the disclosures of which are incorporated herein by reference in their entireties). Particularly suitable technology is such technology, wherein nucleic acid is in a fixed position (such as array or tissue sample) on substrate so that their relative position does not change, and wherein substrate is repeatedly imaged.For example, nucleic acid can be covalently or non-covalently attached to substrate.Such embodiment is particularly suitable, wherein obtains image in different color channels, for example, overlaps with the mark for distinguishing nucleotide base types from each other.In certain embodiments, the method for determining the nucleotide sequence of target nucleic acid can be an automated process.Preferred embodiment comprises synthetic sequencing (" SBS ") technology.

SBS techniques typically involve enzymatic extension of a nascent nucleic acid chain by repeated addition of nucleotides to a template strand. In conventional SBS methods, a single nucleotide monomer can be provided to the target nucleotide in the presence of a polymerase during each delivery. However, in the systems and methods described herein, multiple types of nucleotide monomers can be provided to the target nucleotide in the presence of a polymerase during each delivery.

In some embodiments, the present invention relates to the method for the nucleotide monomer that lacks terminator and the nucleotide monomer that lacks terminator.For example, the method for the nucleotide monomer that lacks terminator comprises pyrophosphate sequencing and uses the order-checking of the nucleotide of gamma-phosphate labeling, as described in further detail below.In the method for the nucleotide monomer that lacks terminator, the number of the nucleotide that adds in each cycle is normally variable, and depends on the pattern that template sequence and nucleotide are sent.For the SBS technology that utilizes the nucleotide monomer with terminator, terminator can be irreversible effectively under the order-checking condition of use, utilizes the situation of traditional Sanger order-checking of dideoxynucleotide, or terminator can be reversible, as the situation of the order-checking method of Illumina, Inc. development.

SBS techniques can utilize nucleotide monomers with or without a label moiety. Accordingly, incorporation events can be detected based on properties of the label, such as fluorescence of the label; properties of the nucleotide monomer, such as molecular weight or charge; byproducts of nucleotide incorporation, such as release of pyrophosphate; and the like. In embodiments where two or more different nucleotides are present in the sequencing reagent, the different nucleotides can be distinguishable from one another, or alternatively, the two or more different labels can be indistinguishable under the detection technology used. For example, different nucleotides present in the sequencing reagent can have different labels, and they can be distinguished using appropriate optical devices, as demonstrated by sequencing methods developed by Illumina, Inc.

Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent chain (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release" Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing" Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time "pyrophosphate" Science 281(5375), 363; U.S. Patent No. 6,210,891; U.S. Patent No. 6,258,568 and U.S. Patent No. 6,274,320, the disclosures of which are incorporated herein by reference in their entireties. In pyrosequencing, the released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via photons generated by luciferase. The nucleic acid to be sequenced can be located on a substrate (e.g., a feature in an array), and the substrate can be imaged to capture the primers resulting from the nucleotides at the positions where the nucleic acid is located on the substrate. In some embodiments, the chemiluminescent signal generated by the addition of a nucleotide sequence is obtained. After using a specific nucleotide type (for example, A, T, C or G) to process the substrate, an image can be obtained. The image obtained after adding each nucleotide type is different in terms of the features detected on the substrate. These differences in the image reflect the different sequence contents of the features on the substrate. However, the relative position of each feature will remain unchanged in the image. The methods described herein can be used to store, process and analyze images. For example, for the images obtained from the different detection channels based on the reversible terminator sequencing method, the images obtained after using each different nucleotide type to process the substrate can be processed in the same manner as demonstrated herein.

In another exemplary type of SBS, cycle sequencing is achieved by progressively adding reversible terminator nucleotides comprising, for example, cleavable or degradable (e.g., photobleachable) dye labels, described in, for example, WO 04/018497 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated herein by reference. This method is being commercialized by Illumina Inc., and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference. Wherein two terminators can be reversed, and the availability of the fluorescently labeled terminator in which the fluorescent marker is cleaved promotes effective cyclic reversible termination (CRT) sequencing. Polymerase can also be co-engineered to effectively incorporate and extend these modified nucleotides.

Preferably, in sequencing embodiments based on reversible terminators, under SBS reaction conditions, the label does not substantially inhibit extension. However, the detection label can be removable, for example, by cleavage or degradation. After the label is incorporated into the nucleic acid features on the array or other substrate, an image can be captured. In a specific embodiment, each cycle involves delivering four different nucleotide types to the substrate simultaneously, and each nucleotide type has a label with a different spectrum. Four images can then be obtained, each image using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, and an image of the substrate can be obtained between each addition step. In such an embodiment, each image will show the nucleic acid features that have been incorporated with a specific type of nucleotide. Due to the different sequence content of each feature, different images will have or not different features. However, the relative position of the features will remain unchanged in the image. The images obtained from such a reversible terminator-SBS method can be stored, processed, and analyzed as described herein. After the image capture step, the label can be removed, and the reversible terminator portion can be removed for subsequent nucleotide addition and detection cycles. Removal of labels (after they have been detected within a particular cycle, and prior to subsequent cycles) can offer the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described below.

In certain embodiments, some or all of the nucleotide monomers may comprise a reversible terminator. In such embodiments, the reversible terminator/cleavable fluorophore may comprise a fluorophore bonded to a ribose moiety via a 3' ester bond (Metzker, Genome Res. 15: 1767-1776 (2005), which is incorporated herein by reference). Other approaches have separated terminator chemicals from the cleavage of fluorescent labels (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety). Ruparel et al. describe the development of a reversible terminator that uses a small 3' allyl group to block extension, but which can be easily unblocked by a short treatment with a palladium catalyst. The fluorophore is connected to the base via a photocleavable linker that can be easily cleaved by exposure to long-wave ultraviolet light for 30 seconds. Therefore, disulfide bond reduction or photocleavage can be used as a cleavable linker. Another method of reversible termination is to use natural termination that occurs after placing a body dye on the dNTP. The presence of a charged body dye on the dNTP can act as an effective terminator through steric hindrance and/or electrostatic hindrance. The presence of an incorporation event prevents further incorporation unless the dye is removed. Cleavage of the dye removes the fluorophore and effectively reverses termination. Examples of modified nucleotides are also described in U.S. Patent No. 7,427,673 and U.S. Patent No. 7,057,026, the disclosures of which are incorporated herein by reference in their entirety.

Additional exemplary SBS systems and methods that can be used with the methods and systems described herein are described in U.S. Patent Application Publication No. 2007/0166705, U.S. Patent Application Publication No. 2006/0188901, U.S. Patent No. 7,057,026, U.S. Patent Application Publication No. 2006/0240439, U.S. Patent Application Publication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, PCT Publication No. WO 06/064199, PCT Publication No. WO 07/010,251, U.S. Patent Application Publication No. 2012/0270305, and U.S. Patent Application Publication No. 2013/0260372, the disclosures of which are incorporated herein by reference in their entireties.

Some embodiments can utilize the detection of four different nucleotides using less than four different labels.For example, the method and system described in the incorporation material of U.S. Patent Application Publication No.2013/0079232 can be used to perform SBS.As a first example, a pair of nucleotide types can be detected under the same wavelength, but based on the intensity difference of a member in the pair and other members, distinguished, or based on the change of a member of the pair (for example, via chemical modification, photochemical modification or physical modification), compared with the detected signal of other members of the pair, it causes obvious signal to appear or disappear.As a second example, three of the four different nucleotide types can be detected under specific conditions, and the fourth nucleotide type lacks a detectable label under these conditions, or is minimally detected under these conditions (for example, due to the minimal detection caused by background fluorescence, etc.).The third nucleotide type is incorporated into nucleic acid and can be determined based on the presence of its corresponding signal, and the fourth nucleotide type is incorporated into nucleic acid and can be determined based on the absence or minimal detection of any signal.As a third example, a nucleotide type can be included in (a plurality of) labels detected in two different channels, and other nucleotide types are detected in no more than one channel. The three exemplary configurations described above are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type detected in both the first and second channels (e.g., dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelengths), and a fourth nucleotide type that lacks a label that is not or minimally detectable in any one channel (e.g., unlabeled dGTP).

Alternatively, sequencing data can be obtained using a single channel, as described in the spiked materials of U.S. Patent Application Publication No. 2013/0079232. In this so-called single-channel sequencing method, a first nucleotide type is labeled, but the label is removed after the first image is generated, and a second nucleotide type is labeled only after the first image is generated. A third nucleotide type retains its label in both the first and second images, and a fourth nucleotide type remains unlabeled in both images.

Some embodiments utilize the sequencing by connecting (ligation) technology. Such technology utilizes DNA ligase to mix oligonucleotide and identify the mixing of such oligonucleotide. Oligonucleotide usually has different labels, which are associated with the identity of the specific nucleotide in the sequence hybridized with the oligonucleotide. Like other SBS methods, it is possible to obtain an image after the processing of the nucleic acid feature on the substrate (for example, array or tissue) of the sequencing reagent with labeling. Each image will illustrate the nucleic acid feature of the labeling having been mixed into a specific type. Due to the different sequence content of each feature, different features will exist or not exist in different images, but the relative position of the feature will remain unchanged in the image. The image obtained from the sequencing method based on connection can be stored, processed and analyzed as described herein. The exemplary SBS system and method that can be used together with the method and system described herein are described in the following documents, U.S. Patent No. 6,969,488, U.S. Patent No. 6,172,218 and U.S. Patent No. 6,306,597, the disclosure of which is incorporated herein by reference in its entirety.

Some embodiments can utilize nanopore sequencing (Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing" Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis" Acc. Chem. Res. 35: 817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J.A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope" Nat. Mater. 2: 611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuations in the pore's electrical conductivity (U.S. Patent No. 7,001,792; Soni, G.V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores" Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis" Nanomed. 2, 459-481 (2007); Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution" J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein. In particular, the data may be processed as images according to the exemplary processing of optical and other images described herein.

Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing polymerase and a gamma-phosphate-labeled nucleotide, as described, for example, in U.S. Patent No. 7,329,492 and U.S. Patent No. 7,211,414 (both of which are incorporated herein by reference), or can be detected using zero-mode waveguides, as described, for example, in U.S. Patent No. 7,315,019 (incorporated herein by reference), and using fluorescent nucleotide analogs and engineered polymerases, as described, for example, in U.S. Patent No. 7,405,281 and U.S. Patent Application Publication No. 2008/0108082 (both of which are incorporated herein by reference). Illumination can be confined to a zeptoliter volume around the surface-tethered polymerase, allowing observation of the incorporation of fluorescently labeled nucleotides with low background (Levene, M.J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations" Science 299, 682-686 (2003); Lundquist, P.M. et al. "Parallel confocal detection of single molecules in realtime" Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures" Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from these methods can be stored, processed, and analyzed as described herein.

Some SBS embodiments include detection of released protons when nucleotides are incorporated into extension products. For example, sequencing based on detection of released protons can use electrical detectors and related technologies commercially available from Ion Torrent (Guilford, CT, Life Technologies subsidiary), or sequencing methods and systems described in the following documents: US 2009/0026082 A1; US 2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, which are incorporated herein by reference. The method described herein for amplifying target nucleic acids using kinetic exclusion can be easily applied to substrates for detecting protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons for detecting protons.

Above-mentioned nucleic acid sequencing method can be advantageously carried out with multiple formats so that a variety of different target nucleic acids are manipulated simultaneously.In a specific embodiment, different target nucleic acids can be processed in common reaction vessels or on the surface of specific substrates.This allows to easily deliver sequencing reagents, remove unreacted reagents, and detect incorporation events in a multiple manner.In the embodiment using surface-bound target nucleic acid, target nucleic acid can be array format.In array format, target nucleic acid can be attached to surface in a spatially distinguishable manner.Target nucleic acid can be combined in the following manner, directly covalently attached, attached to beads or other particles, or attached to polymerase or other molecules attached to surface.Array can comprise a single replication (also referred to as feature) of the target nucleic acid in each site, or can exist multiple replications with identical sequence at each site or feature.Multiple replications can be produced by amplification method, such as bridge amplification or emulsion PCR described in further detail below.

The methods described herein can use arrays having features at any of a variety of densities, including, for example, at least about 10 features/cm ² , 100 features/cm ² , 500 features/cm ² , 1,000 features/cm ² , 5,000 features/cm ² , 10,000 features/cm ² , 50,000 features/cm ² , 100,000 features/cm ² , 1,000,000 features/cm ² , 5,000,000 features/cm ² , or more. Other substrates can contain nucleic acid features at a similar density range.

The advantage of the methods described herein is that they provide rapid and effective detection of multiple target nucleic acids in a parallel manner. Therefore, the present disclosure provides an integrated system that can use technology known in the art to prepare and detect nucleic acids, such as those demonstrated above. Therefore, the integrated system of the present disclosure can include a fluid component that can deliver amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, and the system includes components such as pumps, valves, reservoirs, fluid lines, etc. The flow cell can be configured as and/or used to detect target nucleic acids in the integrated system. Exemplary flow cells are for example described in the following documents, US 2010/0111768 A1 and U.S. Patent No. 8,951,781, which are incorporated herein by reference. As demonstrated by the flow cell, one or more fluid components of the integrated system can be used for amplification method and detection method. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluid components of the integrated system can be used for amplification method as described herein, and for delivering the sequencing reagents in the sequencing method demonstrated above. Alternatively, the integrated system can include a separate fluid system to perform amplification method and perform detection method. Without limitation, examples of integrated sequencing systems capable of generating amplified nucleic acids and determining the sequence of nucleic acids include, but are not limited to, the MiSeq ^™ platform (Illumina, Inc., San Diego, CA), and the apparatus described in U.S. Patent No. 8,951,781, which is incorporated herein by reference.

Confocal imaging system

A confocal TDI line scan imaging system having a high S/N ratio and high confocality to produce high-resolution images is described below with reference to FIG. 1 , FIG. 2 , FIG. 3 , FIG. 4A , FIG. 4B , FIG. 5A , and FIG. 5B .

In certain embodiments, a confocal TDI line scan imaging system includes a detector array that achieves confocality along a scan axis by limiting the scan axis dimension of the detector array. For example, confocality can be achieved along a single axis of the detector array such that confocality occurs only in that dimension. Thus, in contrast to a typical confocal system in which confocality is achieved in two dimensions, a confocal TDI line scan imaging system can be configured such that confocality is not achieved in more than one dimension.

A confocal TDI line scan imaging system can also be configured to sequentially detect different portions of a sample using different subsets of elements of a detector array, where the charge transfer between the subsets of elements occurs at a rate that is synchronized with and in the same direction as the apparent motion of the sample being imaged. For example, a confocal TDI line scan imaging system can scan a sample such that a frame transfer device produces a continuous video image of the sample by stacking linear arrays aligned and synchronized with the apparent motion of the sample, whereby the stored charge moves with it as the image moves from one row to the next. The accumulation of charge can be integrated over the entire time required for the row of charge to move from one end of the detector to a serial register (or, in the case of a frame transfer CCD, to a storage area of the device). An exemplary confocal TDI line scan imaging system is described, for example, in U.S. Patent No. 7,329,860, which is incorporated herein by reference. FIG1 illustrates a side view of an example of a confocal imaging system 100 according to certain embodiments of the present invention. The confocal imaging system is, for example, a TDI line scan imaging system having a high S/N ratio and high confocality to produce high-resolution images.

The presently disclosed confocal imaging system 100 is suitable for use in a photoluminescence-based scanning instrument (or imaging system) for use in a fluorescence-based SBS system.

The confocal imaging system 100 includes a light source aperture 110, a beam splitter 112, a lens 114, a sensor aperture mechanism 130, and a TDI image sensor 146. In the confocal imaging system 100, a tissue sample 120 is arranged at a confocal plane 124 relative to the lens 114. The tissue sample 120 is, for example, a sample tissue to be imaged (or scanned) during an SBS procedure.

The sensor aperture mechanism 130 is positioned in an optically conjugate plane in front of the TDI image sensor 146 to substantially eliminate out-of-focus signals and provide high confocality. That is, various embodiments of the sensor aperture mechanism 130 include a pinhole or slit to substantially eliminate out-of-focus signals. Substantially eliminating out-of-focus signals can be technically advantageous when used for in-situ testing techniques.

As described above, in situ sequencing involves reading sequence information from nucleic acids directly from tissue without extracting the nucleic acids from the tissue. This can be contrasted with sequencing technologies that involve extracting nucleic acids from tissue in order to read sequence information from the extracted nucleic acids. Therefore, in situ sequencing can provide a deeper understanding of the relationship between a cell's genotype or gene expression and its morphology and local environment.

By calibrating the sensor aperture mechanism 130 to substantially eliminate out-of-focus signals, only light from the confocal plane that is focused exactly at the sensor aperture mechanism 130 is allowed to reach the image detector. Thus, the optical resolution of nucleic acids within a specific depth of the tissue can be increased (from the confocal plane that is focused exactly at the slit) relative to a system that does not substantially eliminate out-of-focus signals. This type of optical sectioning simulates the removal of unwanted portions of the tissue (without removing any tissue). In addition, the width of the slit (or the size of the pinhole) may be related to the resolution, with a smaller slit width (or smaller pinhole) providing higher resolution.

In operation, a light source 150 passes through the light source aperture 110, then through the beam splitter 112, then through the lens 114, and impinges on the tissue sample 120 at the confocal plane 124. The light source 150 is an excitation light source used to illuminate the tissue sample 120 during the imaging (or scanning) process. In doing so, the tissue sample 120 emits some focused fluorescence 152 relative to the sensor aperture mechanism 130 and the TDI image sensor 146, as well as some out-of-focus fluorescence 154. The focused fluorescence 152 passes through the sensor aperture mechanism 130 and reaches the TDI image sensor 146, while the out-of-focus fluorescence 154 is rejected by a pinhole or slit in the sensor aperture mechanism 130. In one example, the TDI image sensor 146 is a long linear sensor, such as a 3200×64 pixel sensor, to capture high-resolution images of the tissue sample 120.

2 shows another configuration of the confocal imaging system 100, in which the sensor aperture mechanism 130 is positioned in an intermediate image plane 160 that is conjugate to the TDI image sensor 146. In this configuration of the confocal imaging system 100, an additional pair of lenses 162 is disposed between the sensor aperture mechanism 130 (which is at the intermediate image plane 160) and the TDI image sensor 146. Further details of examples of the sensor aperture mechanism 130 for rejecting out-of-focus light are shown and described below with reference to FIG3 , FIG4A , FIG4B , FIG5A , and FIG5B .

FIG3 illustrates a side view of an example of the sensor aperture mechanism 130 of the confocal imaging system 100 shown in FIG1 and FIG2 . Specifically, FIG3 shows an example of a TDI image sensor 146 comprising a 3200×64 array of pixels 148 (i.e., 3200 columns×64 rows, with the first column being column #1). In this example, the sensor aperture mechanism 130 includes two positionally switchable apertures—one aperture for the odd-numbered columns of the TDI image sensor 146 and another aperture for the even-numbered columns of the TDI image sensor 146. Specifically, the sensor aperture mechanism 130 includes a first aperture plate 132 including slits 134 and a second aperture plate 136 including slits 138. The aperture plates 132 and 136 are formed of a material that is not optically transparent to the wavelengths present in the confocal imaging system 100. For example, the aperture plates 132 and 136 may be formed of a glass substrate coated with a patterned opaque layer (e.g., chromium). Additionally, the height and length of aperture plates 132 and 136 may depend on the overall size of TDI image sensor 146 .

Both aperture plate 132 and aperture plate 136 can be positioned relative to columns of pixels 148 of TDI image sensor 146. The positions of aperture plate 132 and aperture plate 136 are mechanically switchable so that only one of the aperture plates is located in front of TDI image sensor 146 at any given moment. For example, aperture plate 132 and aperture plate 136 can be switchable in a rotational or displacement manner under the control of a controller (not shown). Aperture plate 132 is designed so that, when in front of TDI image sensor 146, the position of slit 134 substantially corresponds to the position of odd-numbered pixel columns of TDI image sensor 146. That is, aperture plate 132 opens to odd-numbered pixel columns of TDI image sensor 146 and blocks even-numbered columns. In contrast, aperture plate 136 is designed so that, when in front of TDI image sensor 146, the position of slit 138 substantially corresponds to the position of even-numbered pixel columns of TDI image sensor 146. That is, aperture plate 136 opens to the even-numbered pixel columns of TDI image sensor 146 and blocks the odd-numbered columns.

In aperture plates 132 and aperture plates 136, placement of slots corresponding to every other (i.e., every second) pixel column ensures adequate rejection of out-of-focus light. Additionally, the sensor aperture mechanism 130 is not limited to just two aperture plates. More than two aperture plates can be used, if desired, to further improve confocality, but at the trade-off of reduced scanning speed. For example, the sensor aperture mechanism 130 can include three aperture plates. A first aperture plate has a slit at the first pixel column, and then at every three pixel columns thereafter. A second aperture plate has a slit at the second pixel column, and then at every three pixel columns thereafter. A third aperture plate has a slit at the third pixel column, and then at every three pixel columns thereafter. To reiterate, the positions of the three aperture plates are mechanically switchable such that only one aperture plate is positioned in front of the TDI image sensor 146 at any given moment.

The slits 134 in the aperture plate 132 and the slits 138 in the aperture plate 136 have a width w. The width w is determined by the size of the pixels 148 of the TDI image sensor 146. In the confocal imaging system 100, the width w of the slits 134 and 138 may be from about 1 μm to about 12 μm in one example, or about 9 μm in another example. The spacing between the slits 134 in the aperture plate 132 and the slits 138 in the aperture plate 136 may depend on the pitch p of the pixels 148 of the TDI image sensor 146. As a non-limiting example, the spacing between the slits 134 in the aperture plate 132 and the slits 138 in the aperture plate 136 may be substantially the same as the pitch p of the pixels 148 of the TDI image sensor 146. Additionally, the lengths of the slits 134 in the aperture plate 132 and the slits 138 in the aperture plate 136 may depend on the overall size of the TDI image sensor 146. As a non-limiting example, the lengths of the slots 134 in the aperture plate 132 and the slots 138 in the aperture plate 136 may be substantially the same as the width of the TDI image sensor 146 along the same dimension of the lengths of the slots 134 and 138 .

The switching cycle of aperture plate 132 and aperture plate 136 is synchronized with the TDI line scan speed, specifically, one switching cycle or an integer number of cycles during TDI scan readout. In operation, during the first imaging or scanning half-cycle, aperture plate 132 is switched to a position in front of TDI image sensor 146, thereby opening the odd-numbered pixel columns of TDI image sensor 146 and blocking the even-numbered pixel columns. During this half-cycle, image data for the odd-numbered pixel columns of TDI image sensor 146 is captured. Then, during the next imaging or scanning half-cycle, aperture plate 132 is switched away, and aperture plate 136 is switched to a position in front of TDI image sensor 146, thereby opening the even-numbered pixel columns of TDI image sensor 146 and blocking the odd-numbered pixel columns. During this half-cycle, image data for the even-numbered pixel columns of TDI image sensor 146 is captured. The movement of aperture plate 132 and aperture plate 136 is synchronized with the high-speed TDI imaging process. In one example, aperture plates 132 and 136 may be switched at a rate of about 5 kHz to about 35 kHz.

Figures 4A and 4B illustrate side views of another example of the sensor aperture mechanism 130 of the confocal imaging system 100 shown in Figures 1 and 2. In this example, only one aperture is used in front of the TDI image sensor 146, where the aperture can be displaced laterally to alternately allow or block odd and even pixel columns. In one example, the aperture plate 132 described with reference to Figure 3 is positioned in front of the TDI image sensor 146 and mechanically displaced laterally during the imaging or scanning process. Figure 4A shows the aperture plate 132 and slit 134 in a first position relative to the TDI image sensor 146, with the odd pixel columns open and the even pixel columns blocked. In contrast, Figure 4B shows the aperture plate 132 and slit 134 in a second position relative to the TDI image sensor 146, with the even pixel columns open and the odd pixel columns blocked.

In operation, during a first imaging or scanning half-cycle, aperture plate 132 is positioned in front of TDI image sensor 146 such that the odd-numbered pixel columns are open and the even-numbered pixel columns are blocked, as shown in FIG4A . During this half-cycle, image data of the odd-numbered pixel columns of TDI image sensor 146 is captured. Then, during the next imaging or scanning half-cycle, aperture plate 132 is mechanically shifted in position in front of TDI image sensor 146 such that the even-numbered pixel columns are open and the odd-numbered pixel columns are blocked, as shown in FIG4B . During this half-cycle, image data of the even-numbered pixel columns of TDI image sensor 146 is captured. Repeating again, the movement of aperture plate 132 can be synchronized with the high-speed TDI imaging process, where the switching rate can be from approximately 5 kHz to approximately 35 kHz.

5A and 5B illustrate side views of yet another example of a sensor aperture mechanism 130 of the confocal imaging system 100 shown in FIG1 and FIG2. In this example, the sensor aperture mechanism 130 is a stationary, electronically controlled spatial light modulator 140. The spatial light modulator 140 can be, for example, a liquid crystal display (LCD)-based device or a microelectromechanical system (MEMS) mirror device. Windows or slits 142 can be electronically disposed in the spatial light modulator 140. The size, number, and position of the windows or slits 142 in the spatial light modulator 140 are electronically controlled.

In the confocal imaging system 100, the spatial light modulator 140 can be used in two states. For example, FIG5A illustrates a first state of the spatial light modulator 140, in which the window or slit 142 is electronically opened and substantially aligned with the odd-numbered columns of pixels 148 of the TDI image sensor 146. In contrast, FIG5B illustrates a second state of the spatial light modulator 140, in which the window or slit 142 is electronically opened and substantially aligned with the even-numbered columns of pixels 148 of the TDI image sensor 146. The switching frequency of the spatial light modulator 140 is synchronized with the high-speed TDI imaging process. In one example, the switching frequency of the spatial light modulator 140 is approximately 5 kHz to approximately 35 kHz. In the confocal imaging system 100, the spatial light modulator 140 is not limited to two states and can be in more than two states.

Focus tracking mechanism during imaging

Certain embodiments of the present invention provide structures that include focus tracking features that can be used to maintain focus during imaging, as described below with reference to Figures 6A, 6B, 7, 8, and 9. For example, the presently disclosed focus tracking mechanisms are suitable for assisting laser-based focusing techniques.

Figures 6A and 6B illustrate, respectively, a plan view and a cross-sectional view of an example structure 600 including focusing strips for improved focus tracking during imaging. In this example, structure 600 includes a bottom substrate 610 and a top substrate 612 with a gap 614 disposed therebetween. Tissue sample 120 can be placed on either bottom substrate 610 or top substrate 612, or both. Bottom substrate 610 and top substrate 612 can be, for example, glass, plastic, or silicon substrates. A set of focusing strips 616 are disposed on the side of top substrate 612 facing gap 614. Focusing strips 616 can be formed, for example, from chromium, gold, or other semiconductor-friendly, highly reflective materials. Focusing strips 616 can be formed on top substrate 612 using standard photolithography processes. Each focusing strip 616 has a thickness t and a width w. In one example, focusing strips 616 have a thickness t of approximately 50 nm and a width w of approximately 50 μm. Focusing strips 616 are arranged at a pitch p. In one example, the pitch p of the focusing stripes 616 is approximately 1100 μm.

In FIG6 and elsewhere herein, the strip shape is exemplified as a reference or light guide. However, it should be understood that other shapes and designs may be used in addition to or as an alternative to the strips. FIG7 illustrates a side view of the structure 600 shown in FIG6A and FIG6B when used in an imaging process. FIG7 illustrates an application that allows imaging through a substrate. FIG7 shows the structure 600 associated with a lens 618 and a lens focused beam 620, wherein the lens 618 and the lens focused beam 620 may be a laser-based focusing mechanism. In this example, imaging is performed through a top substrate 612, and wherein the focusing strips 616 are arranged along the scanning direction (see FIG6A ). The focusing strips 616 are used to assist in focus tracking, wherein each focusing strip 616 has a physical relationship with the tissue sample 120. That is, the focusing strips 616 provide physical features in substantially the same plane of the tissue sample 120 on which the lens focused beam 620 can be focused.

FIG8 illustrates a side view of another example of a structure 600 including a focusing strip 616 for improved focus tracking during imaging. FIG8 illustrates an application that allows imaging without a substrate. In this example, the top substrate 612 is omitted, and the tissue sample 120 is placed on the upper surface of the bottom substrate 610. The focusing strip 616 is disposed on the upper surface of the bottom substrate 610, which abuts the tissue sample 120. The lens 114 and light source 150 used in the fluorescence imaging process are disposed on the exposed side of the tissue sample 120. In contrast, the lens 618 and the laser-based lens focusing beam 620 (as described in FIG7 ) are disposed on the bottom substrate 610 side of the tissue sample 120.

In this configuration, lens 618 and lens focused beam 620, acting as a laser-based focusing mechanism, utilize focusing strips 616 on base substrate 610. A feedback loop is provided from the laser-based focusing mechanism to the fluorescence imaging mechanism. That is, lens 618, lens focused beam 620, and focusing strips 616 are used to generate a focus error signal 630 to the fluorescence imaging mechanism (i.e., lens 114 and light source 150). Focus error signal 630 is used to maintain focus during the imaging (or scanning) process.

9 illustrates a side view of another technique for providing improved focus tracking during imaging. In this example, a tissue sample 120 is placed on top of a base substrate 610, and a strip 122 is cut into the tissue sample 120 to expose a strip of base substrate 610. The exposed strip of substrate can be used as a focusing feature by, for example, a laser-based focusing mechanism (e.g., lens 618 and lens-focused beam 620).

Flow cells for processing tissue samples

Currently, cell culture processes in flow cell chambers are suboptimal. Certain embodiments of the present invention provide flow cells and methods for processing tissue samples, as described below with reference to Figures 10A to 17B.

In certain embodiments, advantageous features of a flow cell for processing tissue include, but are not necessarily limited to, one or more of the following: (1) at least temporary access to a surface of the flow cell that allows a tissue sample to be placed thereon, (2) ease of assembly of flow cell components to at least partially enclose the tissue sample in a flow chamber that allows fluid to contact the tissue sample and allows for formation of a detection area for viewing the tissue sample, and (c) ease of disassembly to allow the tissue sample to be removed for subsequent analysis (e.g., intact tissue or intact portion thereof) or for reuse of the flow cell. In certain embodiments, the integrity of the flow cell will be substantially the same after disassembly and reassembly. In some embodiments, no tools are required for assembly or disassembly. However, in some cases, manual tools may be provided for ease of use rather than requiring the use of power tools.

10A and 10B illustrate a plan view and a cross-sectional view, respectively, of an example of a flow cell 1000 for holding tissue samples and performing any of various types of reaction chemistries (e.g., an SBS chemistry). In this example, the flow cell 1000 includes a bottom substrate 1010 and a top substrate 1012 coupled together using an O-ring 1014. The O-ring 1014 can be formed from fluororubber, silicone, or any other material with process compatibility. That is, the bottom substrate 1010 has a groove 1016 for receiving the O-ring 1014, and the top substrate 1012 has a groove 1018 for receiving the O-ring 1014. When assembled, the O-ring 1014 fits into the groove 1016 of the bottom substrate 1010 and into the groove 1018 of the top substrate 1012, and is sandwiched between the bottom substrate 1010 and the top substrate 1012. The O-ring 1014 is sized so that, when the bottom substrate 1010, the top substrate 1012, and the O-ring 1014 are assembled together, there is a space or gap between the bottom substrate 1010 and the top substrate 1012. In this space or gap, the O-ring 1014 defines a reaction chamber 1020 in the flow cell 1000. In addition, the top substrate 1012 has an inlet 1022 and an outlet 1024 for allowing liquid (e.g., a reagent) to flow into and/or through the reaction chamber 1020 of the flow cell 1000. In one example, the bottom substrate 1010, the top substrate 1012, and the O-ring 1014 can be held together using screws 1026. FIG10B also shows a tissue sample 120 within the reaction chamber 1020 of the flow cell 1000.

Figure 11 illustrates a flow chart of an example of a method 1100 for processing a tissue sample using the flow cell 1000 shown in Figures 10A and 10B. The method 1100 may include, but is not limited to, the following steps.

At step 1110, a first substrate of a flow cell is provided. For example, the bottom substrate 1010 of the flow cell 1000 is provided.

At step 1115 , a tissue sample is placed on a first substrate. For example, tissue sample 120 is placed on the bottom substrate 1010 of flow cell 1000 .

At step 1120, a second substrate is provided and assembled to the first substrate, wherein a reaction chamber is formed around the tissue sample. For example, a top substrate 1012 is provided and assembled to the bottom substrate 1010 using an O-ring 1014 and screws 1026. In doing so, the O-ring 1014 defines a reaction chamber 1020 around the tissue sample 120.

At step 1125, a chemical manipulation is performed on the tissue sample. For example, liquid is flowed into and/or through the reaction chamber 1020 of the flow cell 1000 using the inlet 1022 and the outlet 1024 to perform a chemical manipulation (e.g., an SBS chemical manipulation) on the tissue sample 120. In this example, the imaging or scanning process of the tissue sample 120 can occur through the bottom substrate 1010 and/or the top substrate 1012.

The method can include an imaging step accompanied by sequencing or other nucleic acid detection techniques (such as those described elsewhere herein). Alternatively, the method can include a step of obtaining a picture, image or other representation of the physical form or structure of a tissue sample. The representation can be obtained via light field, fluorescence or other microscopic techniques, and can optionally be assisted by using a dye or a label. The comparison of the representation and the spatially resolved nucleic acid detection results can be used to locate the genetic information with the identifiable characteristics of the tissue. Exemplary methods for spatial detection of nucleic acids that can be modified for use in the apparatus and methods described herein are described in the following documents, U.S. Patent Application Publication No. 2014/0066318A1 and PCT Application Publication No. WO2014/060483A1, both of which are incorporated herein by reference.

Figures 12A and 12B illustrate a plan view and a cross-sectional view of another example of a flow cell 1200 for holding a tissue sample and performing any of various types of reaction chemistry (e.g., SBS chemical process). In this example, the flow cell 1200 includes a bottom substrate 1210 and a top substrate 1212. The bottom substrate 1210 and the top substrate 1212 are combined together using an adhesive layer 1214 sandwiched therebetween. An opening is provided in the adhesive layer 1214 to form a reaction chamber 1216 in the flow cell 1200, the details of which are shown in Figures 14A and 14B. In addition, an inlet 1218 and an outlet 1220 are provided in the top substrate 1212. The inlet 1218 and the outlet 1220 are used to allow a liquid (e.g., a reagent) to flow into and/or flow through the reaction chamber 1216 in the flow cell 1200.

Adhesive layer 1214 is used to couple together bottom substrate 1210 and top substrate 1212. In one example, adhesive layer 1214 is a layer of double-sided tape, such as ultraviolet (UV) cured double-sided tape.

Within the reaction chamber 1216 of the flow cell 1200, the tissue sample can be placed on the top substrate, the bottom substrate, or both substrates. For example, FIG12B shows a tissue sample 120 within the reaction chamber 1216 and on the bottom substrate 1210. In another example, and referring now to FIG13A, the tissue sample 120 within the reaction chamber 1216 is on the top substrate 1212. In yet another example, and referring now to FIG13B, within the reaction chamber 1216, a first tissue sample 120 is on the bottom substrate 1210, and a second tissue sample 120 is on the top substrate 1212.

Reference is now made to Figures 14A and 14B, which are plan and cross-sectional views, respectively, of an example of an adhesive layer 1214 serving as an adhesive portion of the flow cell 1200 shown in Figures 12A and 12B. Specifically, Figures 14A and 14B illustrate openings 1230 in the adhesive layer 1214 that form the reaction chambers 1216 of the flow cell 1200. In one example, the thickness of the adhesive layer 1214 is approximately 100 μm.

Figure 15 illustrates a flow chart of an example of a method 1500 for processing a tissue sample using the flow cell 1200 shown in Figures 12A and 12B. The method 1500 may include, but is not limited to, the following steps.

At step 1510, a first substrate of a flow cell is provided. For example, the bottom substrate 1210 of the flow cell 1200 is provided.

At step 1515 , a tissue sample is placed on a first substrate. For example, the tissue sample 120 is placed on the bottom substrate 1210 of the flow cell 1200 .

At step 1520, a second substrate is provided and then coupled to the first substrate using an adhesive layer, wherein the adhesive layer defines a reaction chamber around the tissue sample. For example, a top substrate 1212 is provided and then coupled to the bottom substrate 1210 using an adhesive layer 1214 (e.g., UV-curable double-sided tape), wherein openings 1230 in the adhesive layer 1214 form a reaction chamber 1216 around the tissue sample 120. In the case of UV-curable double-sided tape, the UV curing operation can occur during this step to form a bond between the adhesive layer 1214 and the bottom substrate 1210 and the top substrate 1212.

At step 1525, a chemical manipulation is performed on the tissue sample. For example, liquid is flowed into and/or through the reaction chamber 1216 of the flow cell 1200 using the inlet 1218 and the outlet 1220, and a chemical manipulation (e.g., an SBS chemical manipulation) is performed on the tissue sample 120. In this example, the imaging or scanning process of the tissue sample 120 can occur through the bottom substrate 1210 and/or the top substrate 1212. Again, imaging can be performed as part of a nucleic acid detection technique and/or to determine the shape or form of the tissue sample.

Figure 16 A and Figure 16B illustrate the side view of the example of the flow cell 1600 that uses open container to keep tissue sample and the example of the process of " dry " imaging of tissue sample therein.In this example, flow cell 1600 comprises open container 1610.Two or more pipes are provided with respect to open container 1610, as its (multiple) inlet and/or (multiple) outlet.For example, pipe 1612 and pipe 1614 are provided with respect to open container 1610, wherein one end of pipe 1612 and one end of pipe 1614 are in open container 1610.That is to say, pipe 1612 and pipe 1614 are used to make liquid 1620 (for example, reagent) flow into and/or flow through open container 1610.In addition, Figure 16 A and Figure 16B show the tissue sample 120 in open container 1610.

FIG16A illustrates the process of imaging a tissue sample 120 in an open container 1610, with the open container 1610 filled with liquid 1620 and the chemical operation occurring on the tissue sample 120. Referring now to FIG16B , upon completion of the chemical operation using tubes 1612 and 1614, the open container 1610 is substantially drained of liquid 1620, and the imaging or scanning process of the tissue sample 120 then occurs through an air gap devoid of liquid 1620. That is, FIG16B illustrates a substantially "dry" imaging process. Some minimal amount of moisture may remain in the open container 1610, such that the tissue sample 120 may not be completely dry.

Referring now to Figures 17A and 17B, a liquid immersion imaging process can be used. For example, Figure 17A shows an open container 1610 filled with liquid 1620 and a chemical operation occurring on a tissue sample 120. The imaging lens (e.g., lens 114) is positioned outside the open container 1610 and is not immersed in the liquid 1620. When the chemical operation is completed, Figure 17B shows the open container 1610 still filled with liquid 1620, and the imaging lens (e.g., lens 114) is lowered into the open container 1610 and immersed in the liquid 1620. In this example, the imaging or scanning process of the tissue sample 120 occurs without an air gap. Without an air gap, resolution and S/N ratio can be improved, and focusing is easier.

In the foregoing detailed description with reference to Figures 1 to 17B, reference is made to the relative positions of components of the structure and/or flow cell (e.g., the relative positions of the top substrate and the bottom substrate of the flow cell), and the terms "top," "bottom," "above," "below," and "upper" are used throughout the description with the understanding that the structures and/or flow cell are functional regardless of their orientation in space.

The above detailed description of the embodiments refers to the accompanying drawings, which show specific embodiments of the present disclosure. Other embodiments with different structures and operations do not depart from the scope of the present disclosure. The terms "invention" and the like are used with reference to certain specific examples of the many alternative aspects or embodiments of the applicant's invention set forth in this specification, and neither their use nor their absence is intended to limit the scope of the applicant's invention or the claims. This specification is divided into two parts only for the convenience of the reader. The title content should not be interpreted as limiting the scope of the invention. The definitions are intended to be part of the description of the invention. It should be understood that various details of the present invention can be changed without departing from the scope of the present invention. In addition, the foregoing description is for illustrative purposes only and not for limiting purposes.

In this application, unless otherwise expressly stated or understood in other ways in the context of use, conditional language such as "may", "can", "possibly" or "can" is generally intended to indicate that certain embodiments include certain features, elements and/or steps, while other embodiments do not. Therefore, such conditional language is generally not intended to imply that features, elements and/or steps are necessary for one or more embodiments in any way, or that one or more embodiments must include logic for determining whether these features, elements and/or steps are included or will be performed in any particular embodiment, with or without user input or prompts. Various publications, patents and/or patent applications have been cited in this application. The entire contents of these publications are incorporated into this application by reference.

The term "comprising" is intended to be open ended and include not only the listed elements, but also any additional elements.

A number of embodiments have been described. However, it should be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the following claims.

The various operations of the above method can be performed by any suitable device capable of performing the operation, such as various hardware and/or software components, circuits, and/or modules. Generally, any operation shown in the figure can be performed by a corresponding functional device capable of performing the operation.

Information and signals may be represented using any of a variety of different technologies. For example, data, instructions, commands, information, signals, bits, symbols, and chips referenced in the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

The various illustrative logic blocks, modules, circuits, and algorithmic steps described in conjunction with the embodiments disclosed herein can be implemented as electronic hardware, computer software, or a combination of the two. In order to clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been generally described in terms of their functionality. Whether such functionality is implemented as hardware or software depends on the specific application and the design constraints imposed on the entire system. The described functionality can be implemented in different ways for each specific application, but such implementation decisions should not be interpreted as resulting in a departure from the scope of the embodiments of the present invention.

The various illustrative blocks, modules, and circuits described in conjunction with the embodiments disclosed herein may be implemented or executed using a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, separate gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general-purpose processor may be a microprocessor, but alternatively, the processor may be any conventional processor, controller, microcontroller, or state machine. The processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

The steps and functions of the methods or algorithms described in conjunction with the embodiments disclosed herein may be implemented directly in hardware, in a software module executed by a processor, or in a combination of the two. If implemented as software, the functions may be stored and transmitted as one or more instructions or codes on a tangible, non-transitory computer-readable medium. The software module may reside in: random access memory (RAM), flash memory, read-only memory (ROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), registers, a hard disk, a removable disk, a CD ROM, or any other form of storage medium known in the art. The storage medium is coupled to the processor so that the processor can read information from the storage medium and write information to the storage medium. Alternatively, the storage medium may be integral to the processor. As used herein, magnetic disks and optical disks include compact disks (CDs), laser disks, optical disks, digital versatile disks (DVDs), floppy disks, and Blu-ray disks, where magnetic disks typically reproduce data magnetically, while optical disks reproduce data optically with lasers. Combinations of the above should also be included within the scope of computer-readable media. The processor and storage medium may reside in an ASIC. The SIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

To summarize the present disclosure, certain aspects, advantages, and novel features of the present invention have been described herein. It should be understood that not all of these advantages may be achieved according to any particular embodiment of the present invention. Accordingly, the present invention may be implemented or practiced in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other advantages that may be taught or suggested herein.

Various modifications to the above-described embodiments will be readily apparent, and the general principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (21)

1. A confocal time-delay integration (TDI) line scan imaging system, comprising: light source; Beam splitter; lens; TDI image sensor; and The TDI image sensor aperture mechanism includes a first aperture plate formed of a material that is not optically transparent to the wavelengths detected by the TDI image sensor. The first aperture plate includes a plurality of slits to focus light onto a plurality of pixel columns of the TDI image sensor, wherein the positions of the plurality of slits correspond to the positions of the plurality of pixel columns of the TDI image sensor. 2. The confocal time-delay integration (TDI) line scan imaging system as described in claim 1, wherein the TDI image sensor aperture mechanism is positioned in an optical conjugate plane in front of the TDI image sensor. 3. The confocal time-delay integration (TDI) line scan imaging system as described in claim 1, wherein the TDI image sensor includes a long linear sensor. 4. The confocal time-delay integration (TDI) line scan imaging system as claimed in claim 1, wherein the TDI image sensor aperture mechanism is positioned in an intermediate image plane conjugate to the TDI image sensor. 5. The confocal time-delay integration (TDI) line scan imaging system as claimed in claim 1, wherein the TDI image sensor aperture mechanism includes a first set of apertures and a second set of apertures having switchable positions. 6. The confocal time-delay integration (TDI) line scan imaging system of claim 5, wherein the first set of apertures is positioned relative to a corresponding first set of pixels on the TDI image sensor, and the second set of apertures is positioned relative to a corresponding second set of pixels on the TDI image sensor. 7. The confocal time-delay integration (TDI) line scan imaging system of claim 1, wherein the TDI image sensor aperture mechanism further includes a second aperture plate, the second aperture plate being formed of a material that is not optically transparent to the wavelength detected by the TDI image sensor, the second aperture plate including a plurality of slits, wherein the positions of the first aperture plate and the second aperture plate are mechanically switchable during the imaging process of the sample. 8. The confocal time-delay integration (TDI) line scan imaging system of claim 7, wherein the plurality of slits of the first aperture plate corresponds to the even-numbered pixel columns of the TDI image sensor, and wherein the plurality of slits of the second aperture plate corresponds to the odd-numbered pixel columns of the TDI image sensor, such that when a switching occurs, at any given moment only one aperture plate is in front of the TDI image sensor. 9. The confocal time-delay integration (TDI) line scan imaging system of claim 7, wherein the controller synchronizes the switching cycle of the first aperture plate and the second aperture plate with the TDI line scan rate of an integer number of cycles in the TDI scan readout. 10. The confocal time-delay integration (TDI) line scan imaging system of claim 9, wherein, in the first imaging half-cycle, the first aperture plate is switched to a position in front of the TDI image sensor, thereby opening the odd-numbered pixel columns of the TDI image sensor and blocking the even-numbered pixel columns of the TDI image sensor. 11. The confocal time-delay integration (TDI) line scan imaging system of claim 9, wherein, in the second imaging half-cycle, the second aperture plate is switched to a position in front of the TDI image sensor, thereby opening the even-numbered pixel columns of the TDI image sensor and blocking the odd-numbered pixel columns of the TDI image sensor. 12. The confocal time-delay integration (TDI) line scan imaging system as claimed in claim 1, further comprising: a substrate on which one or more focusing and tracking mechanisms are disposed. 13. The confocal time-delay integration (TDI) line scan imaging system of claim 12, wherein the focusing tracking mechanism includes a focusing strip. 14. The confocal time-delay integration (TDI) line scan imaging system of claim 13, wherein the focusing strip comprises a highly reflective material. 15. The confocal time-delay integration (TDI) line scan imaging system of claim 12, wherein the focusing and tracking mechanism includes grooves cut into tissue samples. 16. The confocal time-delay integration (TDI) line scan imaging system of claim 15, wherein the groove exposes the surface of the bottom substrate. 17. The confocal time-delay integration (TDI) line scan imaging system of claim 12, wherein the substrate has an exposed side, the exposed side comprising a tissue sample configured to be in direct contact with the focusing tracking mechanism on the same surface as the substrate. 18. The confocal time-delay integration (TDI) line scan imaging system of claim 17, wherein the substrate further comprises a laser-based focusing mechanism disposed on the opposite side of the exposed side. 19. The confocal time-delay integration (TDI) line scan imaging system of claim 1 further includes a flow cell, the flow cell including a first substrate on which a tissue sample to be imaged can be placed. 20. The confocal time-delay integration (TDI) line scan imaging system of claim 19, further comprising a second substrate, wherein the first substrate and the second substrate are separated by a gap, thereby defining a reaction chamber by the first substrate, the second substrate, and the gap. 21. The confocal time-delay integration (TDI) line scan imaging system of claim 20, wherein the flow cell includes an inlet and an outlet for allowing liquid to flow into and through the reaction chamber.