Improved cytometry for organizational characterization and screening

Abstract

To provide imaging systems and methods. [Solution] The optical imaging system includes a frame designed to provide a mechanical coupling between a first stage and a second stage, a sample holding area positioned on the first stage, a lens device, and a sensor array. The lens device is positioned between the first stage and the second stage and is designed to receive light from a sample in the sample holding area of ​​the first stage. The lens device has a numerical aperture of less than 0.1. The sensor array is coupled to the second stage and is designed to receive light passing through the lens device.

Description

Technical Field

[0001] Embodiments of the present invention relate to an imaging system and method, and more particularly to an imaging system that facilitates the analysis and characterization of biological samples, including cells and tissues.

Background Art

[0002] The ability to probe and characterize the expression of molecules on the surface of cells, whether by themselves or in relation to tissues, is essential for researchers to analyze the molecular mechanisms underlying human biology and monitor changes relevant to pathology and therapeutic intervention. In these analyses, it is important to collect information regarding both the types and numbers of molecules expressed on the cell surface and the spatial relationships between cells with different expression profiles. For example, in relation to cancer, spatial information can provide clues as to which cells are inducing an immune response and which structural subgroups are particularly sensitive or resistant to treatment.

[0003] However, state-of-the-art methods for performing such characterization are subject to trade-offs among throughput, parallelization, and maintenance of spatial information. For example, conventional immunofluorescence (IF) and immunohistochemistry (IHC) maintain tissues in a state close to their physiological representation but are limited by the number of spectrally distinct fluorescent probes available and the number of times the tissue can be probed. As a result, one tissue section can only be probed for the expression of 5-7 target molecules. On the other hand, flow cytometry provides the ability to probe 15-18 target molecules. Recent developments in fields such as CyTOF (Cytometry by Time of Flight) utilize antibodies tagged with heavy metal ions and can increase the number of molecules probed to approximately 100. However, in either method, the analysis is performed at the single-cell level, so the original sample needs to be divided and important spatial information is lost. Moreover, once the analysis is performed, the tissue cannot be used for other downstream analyses, such as RNA sequencing, which could potentially provide parallel information. [Overview of the project] [Means for solving the problem]

[0004] In embodiments presented herein, novel imaging techniques are presented to provide a characterization method that preserves spatial information, simultaneously characterizes large tissue fragments, has a theoretically unlimited number of surface molecules that can be probed, and preserves the integrity of the sample for downstream analysis.

[0005] In one embodiment, the optical imaging system includes a frame designed to provide a mechanical coupling between a first stage and a second stage, a sample holding area positioned on the first stage, a lens device, and a sensor array. The lens device is positioned between the first and second stages and is designed to receive light from a sample in the sample holding area of ​​the first stage. The lens device has a numerical aperture of less than 0.1. The sensor array is coupled to the second stage and is designed to receive light passing through the lens device.

[0006] In another embodiment, a method for acquiring a fluorescence image of a sample includes placing the sample on a sample holding area on a stage and placing a substrate containing a plurality of microfluidic channels on the sample. This method further includes flowing a solution through the plurality of microfluidic channels so that the solution comes into contact with the sample and receiving the fluorescence emitted from the sample with a lens device positioned below the stage. The lens device has a numerical aperture of less than 0.1. This method also includes detecting the fluorescence emitted from the sample with a detector positioned optically downstream from the lens device.

[0007] In another embodiment, the optical imaging system includes a frame designed to provide a mechanical coupling between a first stage and a second stage, a sample holding area positioned on the first stage, a substrate positioned on a sample placed in the sample holding area, a lens device, and a sensor array. The substrate includes a plurality of microfluidic channels. The lens device is positioned between the first and second stages and is designed to receive light from the sample in the sample holding area of ​​the first stage. The lens device has a numerical aperture of less than 0.1. The sensor array is coupled to the second stage and is designed to receive light passing through the lens device.

[0008] In another embodiment, a method for detecting a binding reaction comprises sequentially filling a first channel with probe solutions, each of which contains a fluorescently tagged probe molecule. The probe solutions are substantially separated from each other within the first channel. The method further comprises flowing the probe solutions through the first channel to a second channel located on a sample, such that the probe solutions come into contact with the sample when they are present in the second channel. The method comprises using a lens device positioned below the sample to receive fluorescent light from the fluorescently tagged probe molecules present in the sample, and detecting the fluorescent light from the fluorescently tagged probe molecules with a detector positioned optically downstream from the lens device.

[0009] The accompanying drawings incorporated herein and forming part of the specification illustrate embodiments of the invention and, together with the specification, further serve to illustrate the principles of the invention and enable those skilled in the art to construct and utilize the invention. The present invention provides, for example, the following: (Item 1) A frame configured to provide a mechanical connection between the first stage and the second stage, A sample holding area is placed in the first stage, A lens device disposed between the first stage and the second stage, wherein the lens device is configured to receive light from a sample in the sample holding area of ​​the first stage, and the lens device has a numerical aperture of less than 0.1, An optical imaging system comprising: a sensor array coupled to the second stage and designed to receive light passing through the lens device. (Item 2) The optical imaging system according to item 1, wherein the lens device includes a telecentric lens. (Item 3) The optical imaging system according to item 1 or 2, further comprising a housing coupled to the second stage and configured to house the lens device. (Item 4) The optical imaging system according to item 3, wherein the housing further includes one or more of a bandpass filter, a longpass filter, or a polarizing filter. (Item 5) The optical imaging system according to item 3 or 4, wherein the housing includes an opening along the side of the housing, and as a result, light received at the opening is directed towards the sample holding area. (Item 6) The optical imaging system according to item 5, wherein the housing includes an angled filter configured to reflect the light received at the opening and to allow the light received from the sample to pass through. (Item 7) The optical imaging system according to any one of the preceding items, wherein the lens device comprises a plurality of lenses. (Item 8) The optical imaging system according to any one of the preceding items, wherein the sensor array comprises a CMOS sensor array. (Item 9) The optical imaging system according to any one of the preceding items, further comprising a light source configured to provide excitation light toward the sample holding region. (Item 10) The optical imaging system according to any one of the preceding items, wherein the first stage includes one or more bandpass filters, longpass filters, or polarizing filters positioned below the sample holding region. (Item 11) A method for acquiring a fluorescence image of a sample, wherein the method is Placing the sample on the sample holding area on the stage, Placing a substrate containing multiple microfluidic channels on the aforementioned sample, A solution containing fluorescently tagged probe molecules is flowed through the plurality of microfluidic channels so that the solution comes into contact with the sample. The lens device positioned below the stage receives fluorescent light from the fluorescently tagged probe molecule bound to the sample, wherein the lens device receives the fluorescent light having a numerical aperture of less than 0.1. The method comprising detecting the light emitting fluorescence from the fluorescently tagged probe molecule using a detector optically positioned downstream from the lens device. (Item 12) The method according to item 11, further comprising arranging a prism block on the substrate. (Item 13) The method according to item 12, further comprising directing excitation light through the prism block to the sample holding area. (Item 14) The method according to any one of items 11 to 13, wherein the flowing includes flowing the solution through a plurality of microfluidic channels at a substantially constant flow rate. (Item 15) The method according to any one of items 11 to 14, wherein the detection includes detecting the light emitting fluorescence from the tagged biomolecule using a CMOS sensor array. (Item 16) The method according to any one of items 11 to 15, further comprising filtering the light received by the lens device using at least one of a band-pass filter, a long-pass filter, or a polarization filter. (Item 17) The method according to any one of items 11 to 16, wherein the flowing includes flowing the solution through an applied pressure. (Item 18) The method according to any one of items 11 to 17, wherein the lens device includes a telecentric lens. (Item 19) The method according to any one of items 11 to 18, further comprising directing excitation light through the lens device toward the sample holding region. (Item 20) A frame configured to provide a mechanical coupling between a first stage and a second stage, A sample holding region disposed on the first stage, A substrate disposed on a sample placed in the sample holding region, the substrate including a plurality of microfluidic channels, the substrate, A lens device disposed between the first stage and the second stage, the lens device being configured to receive light from the sample in the sample holding region of the first stage, the lens device having a numerical aperture of less than 0.1, the lens device, A sensor array coupled to the second stage and configured to receive light passing through the lens device. An optical imaging system comprising: (Item 21) The optical imaging system according to item 20, wherein the plurality of microfluidic channels includes a plurality of parallel microfluidic channels having lengths oriented in a first direction, wherein the width of each channel of the plurality of parallel microfluidic channels increases with an increase in a distance along a second direction perpendicular to the first direction from a central channel of the plurality of parallel microfluidic channels. (Item 22) The plurality of microfluidic channels includes a network of branched microfluidic channels, where the network of branched microfluidic channels terminates in one fluid channel having a width spanning at least one width of the network of branched microfluidic channels, the optical imaging system according to item 20 or 21 including the plurality of microfluidic channels. (Item 23) A method for detecting a cell binding reaction, the method comprising: sequentially filling a plurality of probe solutions into a first channel, where each of the plurality of probe solutions includes fluorescently tagged probe molecules, and the plurality of probe solutions are substantially separated from each other within the first channel, the filling; sequentially flowing the plurality of probe solutions through the first channel located on the sample into a second channel such that the plurality of probe solutions contact the sample when present in the second channel; using a lens device disposed under the sample to receive light fluorescing from the fluorescently tagged probe molecules present in the sample; detecting, with a detector optically disposed downstream from the lens device, the light fluorescing from the fluorescently tagged probe molecules. The method includes the above. (Item 24) The flowing includes flowing the plurality of probe solutions at a substantially constant flow rate through a plurality of microfluidic channels, the method according to item 23. (Item 25) The detecting includes detecting, with a CMOS sensor array, the light fluorescing from the tagged biomolecules, the method according to item 23 or 24. (Item 26) The method according to any one of items 23 to 25 further includes filtering the light received by the lens device using at least one of a band-pass filter, a long-pass filter, or a polarization filter. (Item 27) The method according to any one of items 23 to 26, wherein the flow includes flowing the plurality of probe solutions through applied pressure. (Item 28) The method according to any one of items 23 to 27, wherein the lens device includes a telecentric lens. (Item 29) The method according to any one of items 23 to 28, wherein the sample is a diseased tissue sample. (Item 30) The method according to any one of items 23 to 29, wherein the sample is a biopsy tissue sample. (Item 31) The method according to any one of items 23 to 30, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises an antibody. (Item 32) The method according to any one of items 23 to 31, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises a protein. (Item 33) The method according to any one of items 23 to 32, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises DNA. (Item 34) The method according to any one of items 23 to 33, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions contains RNA. (Item 35) The method according to any one of items 23 to 34, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises an enzyme. (Item 36) The method according to any one of items 23 to 35, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions contains cells. (Item 37) The method according to any one of items 23 to 36, wherein the fluorescently tagged probe molecules in a given probe solution among the plurality of probe solutions are identical. [Brief explanation of the drawing]

[0010] [Figure 1] A three-dimensional diagram of an optical inspection system according to one embodiment is shown. [Figure 2] A cross-sectional view of an optical inspection system according to one embodiment is shown. [Figure 3] A housing with a lens device, as used in Figure 2, according to one embodiment, is shown. [Figure 4] A cross-sectional view of an optical inspection system according to another embodiment is shown. [Figure 5] Another embodiment shows a housing equipped with a lens device as used in Figure 4. [Figure 6] A top view of a microfluidic apparatus according to one embodiment is shown. [Figure 7] A top view of another microfluidic apparatus according to one embodiment is shown. [Figure 8] A test procedure using a microfluidic apparatus according to one embodiment is shown. [Figure 9A] A and B show exemplary fluorescence measurement values ​​according to one embodiment. [Figure 9B] A and B show exemplary fluorescence measurement values ​​according to one embodiment. [Figure 10] A method for acquiring a fluorescence image of a sample according to one embodiment is shown. [Figure 11] Another method for obtaining a fluorescence image of a sample, according to one embodiment, is shown. [Modes for carrying out the invention]

[0011] Embodiments of the present invention will be described with reference to the accompanying drawings. It should be understood that the drawings are not drawn to a fixed scale, and any specific geometric shapes or dimensions used in the drawings are used solely to provide illustrative embodiments of the present invention.

[0012] While specific configurations and arrangements are described, it should be understood that these are for illustrative purposes only. Those skilled in the art will recognize that other configurations and arrangements can be used without departing from the spirit and scope of the invention. It will be apparent to those skilled in the art that the invention can be used in a variety of applications and is not limited to one specific application.

[0013] References in this specification such as "one embodiment," "embodiment," and "exemplary embodiment" indicate that the described embodiments may include certain features, structures, or characteristics, but it should be noted that not all embodiments necessarily include certain features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiment. Moreover, when certain features, structures, or characteristics are described in relation to an embodiment, it is assumed that achieving such features, structures, or characteristics in relation to other embodiments, whether explicitly stated or not, is within the scope of knowledge of those skilled in the art.

[0014] Traditional microscopy imaging techniques for applications such as intracellular imaging (IF) and intracellular heat conduction (IHC) use high-magnification optical systems to achieve single-cell level resolution, thus sacrificing processing power for high resolution. In these conventional systems, tissue is operationally divided into 30-40 small fragments that fit within the limited field of view of the high-resolution microscope, and each fragment must be imaged independently. As a result, a single tissue sample can take 30 minutes to 2 hours to image, even after the cumbersome processes of fixation, embedding, and labeling (which themselves can take hours or even days). After imaging, further image processing is performed to reconstruct the tissue image from these 30-40 small fragments.

[0015] In contrast, embodiments of this specification relate to optical imaging systems that can be used to acquire images of a sample, where the images are characterized as having a relatively wide field of view without sacrificing resolution. Thus, the entire tissue sample can be imaged in the same field of view (without the need to divide the sample) while maintaining sufficient resolution for both qualitative and quantitative analysis. The sample may include diseased tissue samples or biopsy tissue samples. In some examples, the entire sample is contained within a single image, eliminating the need to take multiple images at different points on the sample.

[0016] Figure 1 shows an optical imaging system 100 according to one embodiment. The optical imaging system 100 includes an upper stage 104 supported by a frame 102. In one embodiment, the frame 102 further provides a mechanical connection between the upper stage 104 and a lower stage 106. The frame 102 may include any number of supports, as shown in Figure 1, or may include other structural shapes or angled members for supporting the upper stage 104 at a given distance above the lower stage 106. The lower stage 106 can provide a stable base for the optical imaging system 100 and may also be connected to a detection element, as will be described in more detail herein. In one embodiment, the upper stage 104 may be designed to move along the Z direction.

[0017] According to one embodiment, the upper stage 104 includes a sample holding area 108. The sample holding area 108 can be located in or near the center of the upper stage 104. The sample holding area 108 can be an opening through the bottom surface of the upper stage 104 so that light can pass through the opening from below or above the upper stage 104. The sample holding area 108 may include a transparent block located on or within the opening. The transparent block may be substantially transparent to all wavelengths of light used during a given cytometry procedure. In some embodiments, the sample holding area 108 may include a recess in the bottom surface of the upper stage 104, including a lower edge for supporting a slide glass or another sample-containing substrate placed in the recess. The sample holding area 108 may further include one or more polarizing filters, bandpass filters, or longpass filters for attenuating excitation light but allowing fluorescence from the sample to pass through.

[0018] According to one embodiment, a housing 110 is positioned between the first stage 104 and the second stage 106. The housing 110 can be coupled to the second stage 106 and can accommodate various optical elements designed to collect light from a sample placed in the sample holding area 108. For example, fluorescence generated by a sample placed in the sample holding area 108 can be collected by the optical elements in the housing 110.

[0019] According to one embodiment, the housing 110 includes a lensing device that receives light from the sample. The lensing device is designed to have a very low numerical aperture so that light can only enter from a narrow range of angles. This helps to significantly improve resolution by eliminating scattered light and other noise sources entering the lensing device. Due to the low numerical aperture, the incidence of light is limited to light propagating in the z direction only, or to small angles from the z direction only. In one embodiment, the numerical aperture of the lensing device in the housing 110 is less than 0.1. In other embodiments, the numerical aperture of the lensing device in the housing 110 is less than 0.05, less than 0.01, or less than 0.001. In one embodiment, the lensing device in the housing 110 does not change the magnification of the acquired image (i.e., the lensing device has a magnification of 1x). In one embodiment, the lensing device includes a telecentric lens. Further discussion of the lensing device is provided in reference to Figures 3A and 3B.

[0020] In one embodiment, the housing 110 includes an opening 112 along the side of the housing 110. Light can be guided into the opening 112 to provide excitation light toward a sample in the sample holding area 108. For example, an optical fiber can be coupled into the opening 112 to guide the excitation light into the opening 112. The excitation light can be used to make the sample in the sample holding area 108 fluorescent.

[0021] In some embodiments, the lower stage 106 may include a removable element 114 that is designed to slide in and out easily. The housing 110 may be coupled to this removable element 114 to provide a mechanism for aligning the housing 110 below the sample holding area 108 (and thus aligning the lens device within it).

[0022] Figure 2 shows side views of various elements of an optical imaging system 100 according to one embodiment. In one example, the sample holding area 108 supports a sample 204. The sample 204 may be placed on a glass slide or other support substrate before being placed on the sample holding area 108. In some embodiments, the sample 204 is a tissue sample.

[0023] In some embodiments, the upper stage 104 includes an optical filter 202 positioned below the sample holding area 108. The optical filter 202 may include one or more of a polarizing filter, a bandpass filter, or a longpass filter. The optical filter 202 may be included when excitation light from an illumination source 210 is directed from above to the sample holding area 108. For example, the illumination source 210 may be a blue laser or a blue LED, and the blue light excites fluorophores in the sample 204 to emit high-wavelength light (e.g., green light). In this scenario, the optical filter 202 can be used to substantially block the passage of blue light while allowing the green light to pass through. For example, a bandpass filter may allow only the band around the green light portion of the electromagnetic spectrum (e.g., about 530 ± 30 nm) to pass through, or a longpass filter may allow only wavelengths above a certain threshold (e.g., above 500 nm) to pass through. Thus, the image of the sample 204 will be formed primarily from the fluorescence of the sample rather than noise (e.g., blue excitation light). Other excitation wavelengths and fluorophore wavelengths can be used in conjunction with the optical filter 202, which is tuned accordingly, to substantially block the passage of excitation light while allowing fluorescence light to pass through.

[0024] In one embodiment, a substrate 206 having one or more microfluidic channels is placed on a sample 204. The substrate 206 may be a glass substrate in which one or more microfluidic channels are etched within the glass substrate. In another embodiment, the substrate 206 is a polymer material such as polydimethylsiloxane (PDMS) molded to form one or more microfluidic channels. The substrate 206 may further include inlet and outlet ports (not shown) for allowing fluid to flow through one or more microfluidic channels. One exemplary arrangement of microfluidic channels in the substrate 206 is shown in Figure 6.

[0025] One or more microfluidic channels can be provided to deliver fluid onto sample 204. Thus, the fluid can come into contact with sample 204 as it flows through one or more microfluidic channels. Tagged probe molecules (e.g., tagged with various fluorophores) in a buffer can be delivered via one or more microfluidic channels to bind to the surface of specific cells or cell types in sample 204. The tagged probe molecules can be cell-binding agents that interact with molecules on or inside the cell surface. Examples of tagged probe molecules include antibodies, proteins, DNA, RNA, enzymes, or cells. According to one embodiment, the binding between the tagged probe molecule and sample 204 is not a covalent bond, but a weak bond based on the equilibrium constant of the binding environment. Because the binding is weak, the flow of solution through one or more microfluidic channels eventually washes away the bound probe molecule. This mechanism allows for the successive introduction of multiple probe molecules onto sample 204 while continuously imaging sample 204 for the binding reactions occurring between any of the tagged probe molecules and sample 204. The introduction of multiple tagged probe molecules will be discussed in more detail later with reference to Figure 7. Various wash buffers can also be delivered through one or more microfluidic channels. In one embodiment, the fluid is delivered through one or more microfluidic channels using a pressure-driven flow.

[0026] In some embodiments, the substrate 206 may be plasma-coupled to a glass slide or other type of glass substrate below the sample 204 using oxygen plasma. For additional leak prevention, an upper substrate 208 may be provided on top of the substrate 206, and downward pressure may be applied to further seal one or more microfluidic channels. In some embodiments, the upper substrate 208 may include one or more screws (not shown) for fastening the upper substrate 208 downward toward the upper stage 104.

[0027] An arbitrary prism block 209 may be provided on the substrate 206. The prism block 209 can be used to reduce the amount of excitation light propagating through the sample holding region 108 toward the housing 110.

[0028] The housing 110 is positioned below the sample holding area 108 to collect light from the sample 204. In one embodiment, the housing 110 includes a lens device and optionally other optical elements for guiding the received light toward the detector 212. The detector 212 may be a sensor array, such as a CMOS sensor array. In one embodiment, the detector 212 can be coupled to the lower stage 106. The detector 212 may have a resolution of, for example, 5 μm. 2 The sensor array may have a pixel size of less than 1.33 mm. Each pixel may have a size of, for example, about 2.2 μm × 2.2 μm. The sensor array may have a total diagonal distance of, for example, less than 8 mm. In one embodiment, the field of view provided by the lens device in the housing 110 is approximately equal to the total footprint of the sensor array when no magnification is imposed by the lens device. Thus, a sensor array with a diagonal distance of 7.33 mm provides an image with a field of view of approximately 7.33 mm along the diagonal.

[0029] Figure 3 shows a diagram of the housing 110 as used in the arrangement shown in Figure 2, according to one embodiment. The housing 110 includes a lens assembly 302, which includes a plurality of lenses 304. The plurality of lenses 304 may include any number and type of lenses, arranged such that the lens assembly 302 has a very small numerical aperture (e.g., a numerical aperture of less than 0.1). In one embodiment, the plurality of lenses 304 include telecentric lenses. Examples of telecentric lenses include both bilateral telecentric lenses and image-side telecentric lenses.

[0030] In some embodiments, the housing 110 includes an optical filter 306. The optical filter 306 may be similar to the optical filter 202 located below the sample holding area 108. Thus, the optical filter 306 may include one or more of a polarizing filter, a bandpass filter, or a longpass filter. In some configurations, it may not be necessary to include both the optical filter 202 and the optical filter 306. Therefore, in some embodiments, only the optical filter 306 is used without the optical filter 202, and in some other embodiments, only the optical filter 202 is used without the optical filter 306.

[0031] Figure 4 shows another diagram of various elements of the optical imaging system 100 according to another embodiment. Many of the same elements shown in Figure 2 are repeated again in Figure 4, and therefore their descriptions will not be repeated here. This embodiment uses a housing 110 with an opening 112 along its side to introduce excitation light. Since the excitation light is provided through the opening 112 and directed from below into the sample holding area 108, the illumination source 210, prism block 209, and optical filter 202, as shown in Figure 2, are not required in this embodiment.

[0032] Figure 5 shows a diagram of the housing 110 as used in the arrangement shown in Figure 4, according to one embodiment. Excitation light 502 is supplied into the housing 110 through an aperture 112. The excitation light 502 is received by an angled filter 506 designed to reflect wavelengths below a threshold and allow wavelengths above a threshold to pass through. According to one embodiment, the excitation light 502 is reflected from the angled filter 506, passes through a lens device 302, and is directed towards the sample holding area 108. Next, light 504 received from the sample is returned to the housing 110 via the lens device 302 and collected, where it passes through the angled filter 506. In some embodiments, the excitation light 502 and light 504 received from the sample do not interact with the same angled filter 506, but instead travel along different optical paths within the housing 110.

[0033] The light 504 received from the sample may include desired fluorescence and / or excitation light 502 mixed with undesirable noise from the surrounding environment. Similar to the embodiment shown in Figure 3, an optical filter 306 may be provided to remove any noise sources from the light 504.

[0034] Figure 6 shows a top view of a flow cell 600 having patterned microfluidic channels according to one embodiment. The flow cell 600 may be an example of the substrate 206 described in relation to Figure 2. The flow cell 600 is placed on a sample (e.g., a tissue sample) and can deliver fluid to a portion of the sample exposed below the microfluidic channels of the flow cell 600.

[0035] According to one embodiment, the flow cell 600 includes two fluid inlet / outlet (I / O) ports 602 and a plurality of parallel microfluidic channels 604 between the I / O ports 602. The plurality of parallel microfluidic channels 604 can be formed using known soft lithography techniques if the flow cell 600 is made of a polymer material. In another example, if the flow cell 600 is made of a more rigid material such as glass or silicon, the plurality of parallel microfluidic channels 604 are etched.

[0036] The fluid is pressure-driven to enter one I / O port 602, and as a result, the fluid flows through each of the multiple microfluidic channels 604 before exiting the opposite I / O port 602. Each of the multiple microfluidic channels 604 can be characterized as having a length l and a width w. According to one embodiment, the length l of each of the multiple microfluidic channels 604 is substantially the same, but the width w of each channel widens as the channel moves away from the central channel 606. For example, the central channel 606 may have the smallest width w, while each of the end microfluidic channels has the largest width w. By varying the width of the microfluidic channels in this pattern, the fluid flows through each of the multiple microfluidic channels 604 at substantially the same flow rate. The width of each of the multiple microfluidic channels 604 can be determined such that the same flow rate is obtained through each channel. This determination may depend on the viscosity of the fluid and the amount of pressure applied to the fluid when it enters the I / O port 602.

[0037] The fluid can flow between the I / O ports 602 using a syringe pump or a pressurized air source. Other forms of fluid transport are also possible, including integrated pumps, capillary action, or electroosmotic flow. As described above, the sample being imaged can form the bottom surface of each of the multiple microfluidic channels 604 so that the fluid flowing through the multiple microfluidic channels 604 flows directly over the sample. By controlling the fluid flow through the multiple microfluidic channels 604, less analyte (compared to non-microfluidic devices) is used in a given experiment with the sample 204. Furthermore, different fluids can be controlledly introduced into different parts of the same sample 204 using other microfluidic designs that utilize multiple separate channels.

[0038] Figure 7 shows a top view of another flow cell 700 having patterned microfluidic channels within it, according to one embodiment. The flow cell 700 may be an example of the substrate 206 described above in relation to Figure 2. The flow cell 700 is placed on a sample (e.g., a tissue sample) and can deliver fluid to a portion of the sample exposed below the microfluidic channels of the flow cell 700.

[0039] According to one embodiment, the fluid enters the flow cell 700 through the inlet port 702 and flows through the inlet channel 704 connected to the inlet port 702. According to one embodiment, the fluid flows from the inlet channel 704 through the branching fluid network 706. As shown in Figure 7, the branching fluid network 706 forms two branching fluid channels at each branching point and branches at three levels, transitioning from one starting channel to eight ending channels. The branching fluid network 706 can contain any total number of branching channels, and any number of branching channels can be used at each branching point.

[0040] According to one embodiment, the branch channels of the branch fluid network 706 terminate in the same large fluid channel 708. The large fluid channel 708 may have a width that spans substantially the entire width of the branch fluid network 706. The large fluid channel 708 may have a width between approximately 15 mm and 25 mm and a length between approximately 20 mm and 30 mm. As the fluid passes through the large fluid channel 708, the fluid flows through the outlet channel 710, which is connected to the fluid outlet 712. According to one embodiment, the branch fluid network 706 spreads the fluid flowing through the inlet channel 704 uniformly along the width of the large fluid channel 708 so that the fluid flows uniformly from end to end of the large fluid channel 708.

[0041] In some embodiments, a flow cell 600 or flow cell 700 is positioned over the sample 204 to deliver fluid to the sample 204. The flow cell 700 may be positioned so that the sample 204 is below a large fluid channel 708. In some embodiments, the fluid flowing through the channel of either the flow cell 600 or flow cell 700 includes a buffer containing a fluorescently tagged probe molecule. The fluorescently tagged probe molecule may bind to a binding partner present on or within the sample 204. The binding partner may be present on or within specific cells or cell types found in the sample 204. The fluorescently tagged probe molecule may include a cell-binding agent that interacts with molecules on or inside the cell surface. The fluorescently tagged probe molecule may include, for example, a fluorescently tagged antibody, protein, DNA, RNA, cell, aptamer, or tissue fragment. A wash buffer may also be flowed through the channel of either the flow cell 600 or flow cell 700 to remove nonspecifically bound molecules from the channel and the surface of the sample 204.

[0042] Figure 8 shows examples of flowing multiple tagged probe molecules through fluid channels on a tissue sample according to several embodiments. The fluid apparatus 800 includes a channel layer 802 on which fluid channels 812 may be patterned. In one example, the channel layer 802 is a glass layer, and the fluid channels 812 are etched into the glass layer. In another example, the channel layer 802 is a polymer layer (e.g., PDMS), and the fluid channels 812 are molded or patterned with the polymer layer. According to several embodiments, the channel layer 802 may have a channel design similar to either the flow cell 600 or the flow cell 700. The fluid apparatus 800 may further include a sample layer 804 containing the sample 805 to be experimented on. The sample layer 804 may be a glass slide or any other transparent material. The sample 805 may be a tissue sample (e.g., a tissue sample obtained during a biopsy of a human or animal subject). In another example, the sample 805 represents an ordered array of biological elements. For example, sample 805 may be an array of DNA sequences, RNA sequences, proteins, enzymes, ligands, antibodies, or any combination thereof. The fluid apparatus may further include a filter layer 806 designed to filter the excitation light and allow the light 816 emitted fluorescently from the tagged probe molecules to pass through.

[0043] The fluid channel 812 can represent any design of a fluid channel for introducing fluid onto the sample 805, and its example in Figure 8 is not intended to be limiting. For example, the fluid channel 812 may include a single channel passing over the sample 805, multiple parallel channels passing over the sample 805, or other more complex configurations that may include branch channels and fluid valves. If the fluid channel 812 includes dimensions less than a millimeter, the fluid channel 812 may be a microfluidic channel.

[0044] According to one embodiment, multiple tagged probe molecules can be delivered sequentially in a continuous arrangement via the fluid channel 812 so that each passes over the sample 805. A series of probe solutions 808-1 to 808-n can be introduced sequentially via the inlet channel 810 so that a series of solution plugs are adjacent to each other. A solution plug may be a defined amount of a particular solution confined within the channel. In another example, each probe solution 808-1 to 808-n may be substantially separated from adjacent probe solutions using an air pocket or buffer. In either case, the probe solutions 808-1 to 808-n are separated from each other by the inlet channel 810 or the fluid channel 812. When the probe solutions 808-1 to 808-n are introduced sequentially as a series of solution plugs, adjacent probe solutions may be substantially separated by a liquid interface. According to one embodiment, diffusion may occur between adjacent solutions across the liquid interface, but no further mixing occurs between the solutions as the solutions flow through at least the inlet channel 810 and the fluid channel 812. The lack of liquid interface formation between solutions and substantial mixing can occur due to the small shape of the microfluidic channels, which provide laminar flow of solutions through them.

[0045] Probe solutions 808-1 to 808-n may each contain the same group of probe molecules. The same group of probe molecules may differ between different probe solutions 808-1 to 808-n.

[0046] In the example shown in Figure 8, probe solution 808-1 is initially introduced onto sample 805 for a certain period of time based on various factors. These factors may include the dimensions of the fluid channel 812, the flow rate of the solution, and the amount of probe solution 808-1 filled. Those skilled in the art will understand how to adjust one or more of these factors to affect the duration of time the probe solution is present on the sample. After probe solution 808-1 has finished flowing over sample 805, it flows out through the outlet channel 814, followed immediately or after a set period by probe solution 808-2, etc., until each of probe solutions 808-1 to 808-n has been introduced onto sample 805.

[0047] Fluorescence 816 from the tagged probe molecule is collected as the molecule flows over sample 805. Figures 9A and 9B show examples of fluorescence signals collected over time for a scenario where no binding occurs (A) and a scenario where binding occurs (B). For clarity, Figures 9A and 9B do not include the collected excitation light, which would be present to some extent in the measured optical signal. Furthermore, relative peak sizes and widths are shown for illustrative purposes only and should not be considered limiting.

[0048] If no binding occurs, the probe solution flows over sample 805, and the fluorescent tag remains on the optically collected area for only a short time (because it does not bind to any part of sample 805). Therefore, the resulting fluorescence signal appears as a single sharp peak centered on the peak emission wavelength of the fluorophore. However, if the probe molecules in the probe solution show some degree of binding to any part of sample 805, the fluorescent tag will remain on the optically collected area for a longer period. This appears as a fluorescence signal that is further drawn out over time, as shown in Figure 9B. The slow decay of the collected fluorescence signal occurs because the fluorescently tagged probe molecules are slowly washed away from their binding site as the fluid continues to flow in the channel. The rate of decay of the fluorescence signal may be affected by the flow rate of the solution or the amount of probe solution packed in.

[0049] In some embodiments, the characteristics of the fluorescence signal collected from the probe molecule can be analyzed to determine whether binding occurs, which can then be used to determine whether a particular cell type or biological entity is present in sample 805. In some embodiments, the characteristics of the fluorescence signal collected from the probe molecule can be analyzed to determine the concentration or total number of target molecules (e.g., molecules that bind to the probe molecule) present in sample 805. In some embodiments, multiple emission peaks may be present simultaneously, corresponding to different fluorophores on different probe molecules.

[0050] Figure 10 shows a flowchart of Method 1000 for acquiring a fluorescence image of a sample according to one embodiment. Various steps of Method 1000 can be performed using embodiments of the optical imaging system 100 described herein. It should be understood that other steps may occur between the steps shown herein, but are omitted for clarity and brevity. Such steps would include conventional sample preparation techniques, which would be well understood by those skilled in the art.

[0051] Method 1000 begins with step 1002, in which a tissue sample is placed on a sample holding area. At least a portion of the sample holding area may be transparent to substantially all wavelengths of light used during a given cytometry procedure. In some embodiments, the tissue sample is first placed on a glass slide (or a similar transparent substrate) before being placed on the holding area. The glass slide may fit into a recess in the sample holding area.

[0052] Method 1000 follows step 1004, in which a substrate having microfluidic channels is placed on the sample. The substrate may be a glass substrate in which the microfluidic channels are etched within the glass substrate. In another embodiment, the substrate is a polymer material such as PDMS molded to form the microfluidic channels. Other components may also be added to the substrate. For example, a transparent prism block may be placed on the substrate to direct the excitation light toward the prism block. The prism block may be included to reduce the amount of excitation light received by a downstream lensing device.

[0053] Method 1000 follows step 1006, in which a fluid flows through microfluidic channels (or more) in the substrate. For a substrate containing multiple microfluidic channels, the fluid can flow through each of the multiple parallel microfluidic channels at substantially the same flow rate. The flow rate can be determined based on the shape of the microfluidic channels (or more). The fluid flow can be controlled via an applied pressure supplied by a syringe pump or pressurized air.

[0054] Method 1000, following step 1008, according to one embodiment, involves receiving fluorescent light from tagged probe molecules present in the sample (e.g., bound to or passing through the sample) in a lens device positioned beneath the sample holding area. The lens device may be characterized as having a very small numerical aperture (e.g., less than 0.1). In other embodiments, the numerical aperture of the lens device is less than 0.05, less than 0.01, or less than 0.001. In one example, the lens device includes a telecentric lens. In some embodiments, excitation light is directed through the lens device to the sample holding area, while fluorescent light from the sample is collected through the lens device. The lens device may be located within a housing.

[0055] Method 1000 follows step 1010, in which light received by a lens device is detected using a sensor array. The sensor array may include a CMOS array of detectors. In one embodiment, the field of view provided by the lens device is approximately equal to the total footprint of the sensor array, if no magnification is imposed by the lens device. Thus, the physical size of the sensor array in such embodiments is approximately equal to the field of view of the acquired image. For example, a sensor array with a diagonal distance of 7.33 mm provides an image with a field of view of approximately 7.33 mm along the diagonal. In some embodiments, the light received by the lens device is filtered before it is received by the sensor array. Filtering may be performed by one or more of bandpass filters, longpass filters, or polarizing filters. In some embodiments, the filtering of the light is performed before the light is received by the lens device.

[0056] In some embodiments, light received by a sensor array can be used to form an image of the sample. This image may provide detail of areas of the sample stained with tagged probe molecules (e.g., fluorescently tagged probe molecules). The image may further provide detail of areas of the sample where binding has occurred between the sample and the fluorescently tagged probe molecules. The intensity of the fluorescent areas in the image can be used to indicate the degree of binding that has occurred. For example, an image showing bright areas of the sample may indicate a high level of binding between the fluorescently tagged probe molecules and the sample in those bright areas.

[0057] In some embodiments, the light received by the sensor array includes any fluorescence generated from fluorescently tagged probe molecules in the sample. Using the intensity of this received fluorescence, the concentration of fluorescently tagged probe molecules present in the sample can be calculated. Such experiments may be performed, for example, to determine the relative concentrations of a particular cell type, a specific protein on the outer surface of a cell, or any other biomolecules present in the sample. In some embodiments, light is received from the entire sample, resulting in the generation of a single image of the entire sample region.

[0058] Figure 11 shows a flowchart of Method 1100 for high-throughput analysis of multiple tagged probe molecules according to one embodiment. Various steps of Method 1100 can be performed using embodiments of the optical imaging system 100 and / or fluid apparatus 800 described herein. It should be understood that other steps may occur between the steps shown herein but have been omitted for clarity and brevity. Such steps would include conventional sample preparation techniques, which would be well understood by those skilled in the art.

[0059] Method 1100 begins with step 1102, in which a tissue sample is placed on a sample holding area, followed by step 1104, in which a substrate having microfluidic channels is placed on the sample. These steps are similar to steps 1002 and 1004 already described in relation to Method 1000, and therefore their descriptions will not be repeated here.

[0060] Method 1100 follows step 1106 in which a series of solutions containing tagged probe molecules are filled into channels connected to a microfluidic channel(s). The probe solutions can be sequentially filled into syringes or plastic tubes that ultimately lead to the microfluidic channel(s). The probe solutions may be filled so that each probe solution comes into contact with adjacent probe solutions in the channel (e.g., to form a liquid interface) as they flow together through the channel. In another embodiment, the probe solutions may be filled so that there is space between each probe solution in the channel. The space may be filled with air or another solution such as a buffer.

[0061] Method 1100 follows step 1108, in which fluorescently tagged probe molecules flow over a tissue sample. The flow can be pressure-driven, allowing the probe molecules to flow continuously over the tissue sample. The flow rate can be adjusted to vary the time each probe solution remains on the tissue sample. The time a given probe solution remains on the sample can be approximately seconds (e.g., between 10 and 30 seconds). High-throughput detection of multiple binding activities can be achieved by sequentially flowing any number of fluorescently tagged probe molecules over the entire tissue sample.

[0062] Method 1100 follows step 1110, in which fluorescent light from tagged probe molecules present in the sample is received by a lens device positioned beneath the sample holding area. Next, Method 1100 follows step 1112, in which the light received by the lens device is detected using a sensor array. These steps are similar to steps 1008 and 1010 already described in relation to Method 1000, and therefore their descriptions will not be repeated here.

[0063] lastly It should be understood that the section describing embodiments for carrying out the invention, rather than the section describing the summary and abstract of the invention, is intended to be used to interpret the claims. The section describing the summary and abstract of the invention may describe one or more, but not all, exemplary embodiments of the invention as intended by the inventor(s) and therefore does not in any case limit the scope of the invention and the appended claims.

[0064] Embodiments of the present invention have been described above using functional building blocks that demonstrate the realization of their specific functions and relationships. The boundaries of these functional building blocks are arbitrarily defined herein for descriptive convenience. Other boundaries may be defined as long as their specific functions and relationships are adequately performed.

[0065] The above description of specific embodiments fully discloses a general feature of the invention that, by applying knowledge within the scope of the art, others may readily modify and / or adapt such specific embodiments for various applications without excessive experimentation and without departing from the general concept of the invention. Therefore, such modifications and adaptations are intended to fall within the meaning and scope of the equivalents of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the language or terminology herein is for illustrative purposes only and not for limitation, so that it should be understood by those skilled in the art in light of the teachings and guidance.

[0066] The effects and scope of the present invention should not be limited by any of the exemplary embodiments described above, but should be defined solely by the following claims and their equivalents.

Claims

1. A method for detecting a cell binding reaction, wherein the method is: The method involves sequentially filling a first channel with multiple probe solutions, wherein each of the multiple probe solutions contains a fluorescently tagged probe molecule, and the multiple probe solutions are substantially separated from each other within the first channel. The plurality of probe solutions are sequentially flowed through the first channel to the second channel located on the sample, so that the plurality of probe solutions come into contact with the sample when they are present in the second channel. The lens array placed beneath the sample is used to receive fluorescence emitted from the fluorescently tagged probe molecules present in the sample. A detector optically positioned downstream from the lens array detects the fluorescence emitted from the fluorescently tagged probe molecule. Methods that include...

2. The method according to claim 1, wherein the flowing comprises flowing the plurality of probe solutions through a plurality of microfluidic channels at a substantially constant flow rate.

3. The method according to claim 1, wherein the detection includes detecting the fluorescence emitted from the fluorescently tagged probe molecule with a CMOS sensor array.

4. The method according to claim 1, further comprising filtering the light received by the lens array using at least one of a bandpass filter, a longpass filter, or a polarizing filter.

5. The method according to claim 1, wherein the flow includes flowing the plurality of probe solutions through applied pressure.

6. The method according to claim 1, wherein the lens array includes a telecentric lens.

7. The method according to claim 1, wherein the sample is a diseased tissue sample.

8. The method according to claim 1, wherein the sample is a biopsy tissue sample.

9. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises an antibody.

10. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions contains a protein.

11. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions contains DNA.

12. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises RNA.

13. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions comprises an enzyme.

14. The method according to claim 1, wherein the fluorescently tagged probe molecule in one or more of the plurality of probe solutions contains cells.

15. The method according to claim 1, wherein the fluorescently tagged probe molecules in a given probe solution among the plurality of probe solutions are identical.

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