Patent Description:
This disclosure relates generally to surface plasmon resonance (SPR) imaging systems, and methods to use such systems, for measuring molecular interactions.

Surface plasmon resonance (SPR) detection using incident light beam is a popular technique for monitoring molecular interactions in real-time. However, traditional SPR devices or systems are not suitable for the study of heterogeneity effects naturally occurred in cell population because they either have limited fields of view or are not design for imaging cellular structures or phenotypes that often have random patterns and structures. Therefore, systems and methods configured to have a large field of view and a high resolution for measuring molecular interactions in real time are desired. <CIT> describes a multi-channel surface plasmon resonance sensor using beam profile ellipsometry; and, more particularly, to a high sensitive measuring technology, which is coupled with a vertical illumination type focused-beam ellipsometer using a multi-incident angle measurement method, and a surface plasmon resonance (SPR) sensing part deposited with a metal thin film. The multi-channel surface plasmon resonance sensor includes a vertical illumination type focused-beam ellipsometer, in which light is polarized; a surface plasmon resonance (SPR) sensing part which is provided at the objective lens part of the focused-beam ellipsometer so as to generate SPR according to an angle change of the polarized light; and a flow unit which supplies a buffer solution containing a bio material binding to or dissociation from the metal thin film generating surface plasmon, wherein the SPR and the ellipsometric phase change by change in an angle and a wavelength are simultaneously detected. <CIT> describes a full-automatic surface plasmon resonance (SPR) biological analyzer which comprises an SPR sensor, an integrated micro-flow chuck in fit with the SPR sensor, a full-automatic sampling system used for providing a sampling source for the SPR sensor, a constant temperature control cabinet used for carrying out constant temperature control on the SPR sensor and the integrated micro-flow chuck, a system communication controller serving as a communication center, and an upper computer which is used for receiving signals of the SPR sensor and controlling the work of the full-automatic sampling system and the constant temperature control cabinet. <CIT> describes a measuring instrument utilizing surface plasmon resonance. The instrument employs no moving mechanical parts and produces the resonance by changing the parameters either electrically or acoustically, and it detects the reflection by a stationary detector. <CIT> describes a surface plasmon resonance imaging sensor capable of performing absolute calibration comprising: a transparent substrate; a first prism and a second prism formed at one surface of the substrate and symmetrically positioned with reference to the center axis of the substrate; an optical system for providing light to the first and second prisms; and a light receiving part for detecting the light reflected from the substrate, wherein a surface plasmon resonance (SPR) angle change of an object to be measured by the first prism is measured, and a refractive index change on each pixel of the object is obtained as a two-dimensional difference image by the second prism. <NPL>) describes i) using an optical block as a coupler, the incident beam falling on it in two perpendicular directions, or ii) the incident beam being directly launched into a sample from a substrate side, with a thin metal film deposited on the substrate and covered by either air or another dielectric layer.

The invention is defined by the independent claims, and preferred embodiments are set out in the dependent claims.

To facilitate further description of the embodiments, the following drawings are provided in which:.

For simplicity and clarity of illustration, the drawing figures illustrate the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the present disclosure. Additionally, elements in the drawing figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of embodiments of the present disclosure. The same reference numerals in different figures denote the same elements.

The terms "first," "second," "third," "fourth," and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "include," and "have," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, device, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, system, article, device, or apparatus.

It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the apparatus, methods, and/or articles of manufacture described herein are, for example, capable of operation in other orientations than those illustrated or otherwise described herein.

The terms "couple," "coupled," "couples," "coupling," and the like should be broadly understood and refer to connecting two or more elements mechanically and/or otherwise. Two or more electrical elements may be electrically coupled together, but not be mechanically or otherwise coupled together. Coupling may be for any length of time, e.g., permanent or semi-permanent or only for an instant. "Electrical coupling" and the like should be broadly understood and include electrical coupling of all types. The absence of the word "removably," "removable," and the like near the word "coupled," and the like does not mean that the coupling, etc. in question is or is not removable.

As defined herein, two or more elements are "integral" if they are comprised of the same piece of material. As defined herein, two or more elements are "non-integral" if each is comprised of a different piece of material.

As defined herein, "approximately" can, in some embodiments, mean within plus or minus ten percent of the stated value. In other embodiments, "approximately" can mean within plus or minus five percent of the stated value. In further embodiments, "approximately" can mean within plus or minus three percent of the stated value. In yet other embodiments, "approximately" can mean within plus or minus one percent of the stated value.

As defined herein, "real-time" can, in some embodiments, be defined with respect to operations carried out as soon as practically possible upon occurrence of a triggering event. A triggering event can include receipt of data necessary to execute a task or to otherwise process information. Because of delays inherent in transmission and/or in computing speeds, the term "real time" encompasses operations that occur in "near" real time or somewhat delayed from a triggering event. In a number of embodiments, "real time" can mean real time less a time delay for processing (e.g., determining) and/or transmitting data. The particular time delay can vary depending on the type and/or amount of the data, the processing speeds of the hardware, the transmission capability of the communication hardware, the transmission distance, etc. However, in many embodiments, the time delay can be less than approximately one second, five seconds, ten seconds, thirty seconds, one minute, five minutes, ten minutes, or fifteen minutes.

Turning to the drawings, <FIG> illustrate various views of an SPR imaging system <NUM>, according to an embodiment. In this and other embodiments, the SPR imaging system <NUM> can comprise: (a) an optical assembly <NUM>; (b) an SPR light source <NUM>; (c) an SPR camera <NUM>; (d) a bright field light source <NUM>; and/or (e) a bright field camera <NUM>. In many embodiments, the SPR imaging system <NUM> can comprise a high optical resolution, such as an optical resolution not larger than <NUM> micrometers (µm), <NUM>, etc. In these and other embodiments, the SPR imaging system <NUM> also can comprise a wide SPR angle, such as an SPR angle ranging from <NUM> to <NUM> degrees. In some embodiments, the SPR imaging system <NUM> can further comprise a large optical field of view, such as an optical field of view as great as <NUM> millimeters-squared (mm<NUM>), <NUM><NUM>, or <NUM><NUM>. According to the invention, the SPR imaging system <NUM> is configured to simultaneously capture or process an SPR image and a bright field image in real time.

In many embodiments, the optical assembly <NUM> of the SPR imaging system <NUM> can comprise: (a) a hemispherical prism <NUM> that comprises a planar top surface <NUM> configured to support a surface-plasmon-resonance (SPR) sensor <NUM>; (b) a high numerical aperture (NA) lens <NUM>; and/or (c) a housing <NUM> configured to mount the hemispherical prism <NUM> and the high NA lens <NUM>, such that the high NA lens <NUM> is located distal from the planar top surface <NUM> of the hemispherical prism <NUM>. In some embodiments, the top surface of the hemispherical prism is not planar or is not entirely planar.

In many embodiments, with the hemispherical prism <NUM> configured to support the SPR sensor <NUM>, no sensor supporting stage is needed, and the SPR imaging system <NUM> thus can have fewer heat leaking surfaces and fewer sources of mechanical vibration noise. In many embodiments, the hemispherical prism <NUM> can comprise a high refractive index, such as a refractive index no less than <NUM>. In these and other embodiments, the high NA lens <NUM> can comprise a radius at least <NUM> times greater than a radius of the hemispherical prism <NUM>. In some embodiments, the high NA lens <NUM> can comprise a high NA value, such as no less than <NUM>.

In many embodiments where optical assembly <NUM> of the SPR imaging system <NUM> comprises the housing <NUM>, the hemispherical prism <NUM> and the high NA lens <NUM> can be configured to be firmly coupled to the housing <NUM> in order to eliminate any relative movement between the hemispherical prism <NUM> and the high NA lens <NUM> that can cause mechanical vibration noises. In these and other embodiments, the housing <NUM> can enclose at least a portion of each of the hemispherical prism <NUM> and the high NA lens <NUM> for better temperature control. In one such embodiment, the housing <NUM> can enclose the area between the hemispherical prism <NUM> and the high NA lens <NUM>. In the same or different embodiment, the housing <NUM> can leave the planar top surface <NUM> of the hemispherical prism <NUM> and the bottom surface of the high NA lens exposed, as in the exemplary embodiment shown in <FIG> and <FIG>.

In many embodiments, the SPR light source <NUM> of the SPR imaging system <NUM> can be configured to emit a low-coherent monochromatic light beam <NUM> for SPR imaging toward the high NA lens <NUM>. In many embodiments, an incident angle of the low-coherent monochromatic light beam <NUM> from the SPR light source <NUM> towards the high NA lens <NUM> can be adjustable, such as by one or more additional optical components or by adjusting the location or angle of the SPR light source <NUM>. In many embodiments, the SPR camera <NUM> can be configured to capture an SPR image formed after the low-coherent monochromatic light beam <NUM> is incident upon and reflected by a metal-coated sample contacting surface <NUM> of the SPR sensor <NUM>.

According to the invention, the SPR imaging system <NUM> comprises the bright field light source <NUM> and the bright field camera <NUM> for bright field imaging. In many embodiments, the bright field light source <NUM> can be configured to emit a bright field light beam <NUM> to illuminate the metal-coated sample contacting surface <NUM> of the SPR sensor <NUM>, and the bright field camera <NUM> can be configured to capture a bright field image of the SPR sensor <NUM>. In some and other embodiments, the bright field light source <NUM> can additionally comprise a condenser. As an example, the condenser can be configured to render a light beam, that is emitted from the bright field light source <NUM> and originally divergent, into a parallel and/or convergent bright field light beam to illuminate the SPR sensor <NUM>. In many embodiments, the SPR imaging system <NUM> can be configured so that the bright field camera <NUM> can capture the bright field image of the SPR sensor <NUM> simultaneously with the SPR camera capturing the SPR image.

In many embodiments, the high NA lens <NUM> can be configured to refract the low-coherent monochromatic light beam <NUM> from the SPR light source <NUM> toward the hemispherical prism <NUM>. In the same or different embodiments, the high NA lens <NUM> can condition the low-coherent monochromatic light beam <NUM>. As an example, the conditioning provided by the high NA lens <NUM> can bend the refracting beam <NUM>-<NUM> degrees from incident beam path, for both beams towards and away from the sensor. In many embodiments, the hemispherical prism <NUM> can be configured to collimate the low-coherent monochromatic light beam <NUM>, as refracted by the high NA lens <NUM>, toward the SPR sensor <NUM> that is coupled to the planar top surface <NUM> of the hemispherical prism <NUM>.

In many embodiments, the SPR camera <NUM> and/or the bright field camera <NUM> can be communicably coupled to a computing device <NUM> (<FIG>), such as a computer or a server, that is configured to: receive and record the SPR image from the SPR camera; receive and record the bright field image from the bright field camera; calibrate the SPR image from the SPR camera; calibrate the bright field image from the bright field camera; and map the SPR image onto the bright field image for binding analysis. In some embodiments, the computing device <NUM> (<FIG>) can be further configured to automatically perform one or more data processing procedures, including: (a) normalizing the SPR sensitivity of the sensor surface per pixel and removing the sensor inhomogeneity by one or more calibration techniques, such as an SPR profile scan, injection of a known index standard, a thermal response, or another known method to determine localized SPR sensor sensitivity; (b) removing sensor drift by leveling the baseline with reference, linear, and non-linear subtractions; (c) identifying anomalous data of binding behaviors and eliminating artifacts in the data by using Artificial Intelligence ("AI") to categorize binding behaviors and exclude the data which do not match between isotherm analysis and kinetic analysis; and/or (d) identifying one or more sets of data showing model behaviors as the model data and analyzing the model data to derive a measured result with a certain confidence level.

Furthermore, in many embodiments, the SPR imaging system <NUM> can comprise additional optical components, such as one or more lenses, one or more mirrors, a phase-shift ring <NUM> (<FIG>), and/or a color filter <NUM> (<FIG>), and so forth, for adjusting the colors or directions of the light beams and/or the colors or contrast of the bright field image or the SPR image for SPR imaging, bright field imaging, phase-contrast imaging, and/or fluorescence imaging. For instance, in some embodiments, the phase-shift ring <NUM> can be configured for phase-contrast imaging with the bright field light source <NUM> and the bright field camera <NUM> to increase the contrast of the bright field image, and the phase-shift ring <NUM> can be located in any suitable place, such as between the hemispherical prism <NUM> and the high NA lens <NUM> or between the optical assembly <NUM> and the bright field camera <NUM>. In many embodiments, the bright field light source <NUM> and the bright field camera <NUM> can be further configured for fluorescence imaging with one of: (a) the bright field light beam <NUM> emitted by the bright field light source <NUM> comprising a colored light; or (b) the bright field light beam <NUM> emitted by the bright field light source <NUM> comprising a white light and the color filter <NUM> located at one of: between the bright field light source <NUM> and the SPR sensor <NUM> or in front of bright field camera <NUM>, and configured to change one or more colors of bright field light beam <NUM>.

Turning to the drawings, <FIG> illustrates an optical assembly <NUM>, according to an embodiment. In many embodiments, the optical assembly <NUM> can comprise a hemispherical prism <NUM>, a high NA lens <NUM>, and/or a housing <NUM>. In many embodiments, the hemispherical prism <NUM> can comprise a planar top surface <NUM> configured to support an SPR sensor <NUM> with a metal-coated sample contacting surface <NUM>. In some embodiments, the top surface of the hemispherical prism is not planar or is not entirely planar. In many embodiments, the housing <NUM> can be configured to mount the hemispherical prism <NUM> and the high NA lens <NUM>, such that the high NA lens <NUM> is located distal from the planar top surface <NUM> of the hemispherical prism <NUM>. In many embodiments, an SPR imaging system adopting the optical assembly <NUM> can comprise: a high optical resolution, such as an optical resolution not larger than <NUM>, <NUM>, etc.; a wide SPR angle, such as an SPR angle ranging from <NUM> to <NUM> degrees; and/or a large optical field of view, such as an optical field of view as great as <NUM><NUM>, <NUM><NUM>, or <NUM><NUM>. According to the invention, the optical assembly <NUM> is used for simultaneous SPR and bright field imaging.

In many embodiments, the hemispherical prism <NUM> can comprise a high refractive index, such as a refractive index no less than <NUM>. In these and other embodiments, the high NA lens <NUM> can comprise a radius at least <NUM> times greater than a radius of the hemispherical prism <NUM>. In some embodiments, the high NA lens <NUM> can comprise a high NA value, such as no less than <NUM>. In many embodiments, the hemispherical prism <NUM> and the high NA lens <NUM> can be configured to be firmly coupled to the housing <NUM> in order to eliminate any relative movement between the hemispherical prism <NUM> and the high NA lens <NUM> that can cause mechanical vibration noise. In these and other embodiments, the housing <NUM> can enclose at least a portion of each of the hemispherical prism <NUM> and the high NA lens <NUM> for better temperature control, such as enclosing the area between the hemispherical prism <NUM> and the high NA lens <NUM>.

Turning to the drawings, <FIG> illustrate various views of an SPR imaging system <NUM>, according to another embodiment. In this and other embodiments, the SPR imaging system <NUM> can comprise: (a) an optical assembly <NUM>; (b) an SPR light source <NUM>; (c) an SPR camera <NUM>; (d) a bright field light source <NUM>; (e) a bright field camera <NUM>; (f) a thermoelectric device <NUM>; (g) a microfluidic device <NUM>; and/or (h) a sensor translation mount <NUM> (<FIG>). In many embodiments, the optical assembly <NUM> can comprise a hemispherical prism <NUM>, a high NA lens <NUM>, a thermoelectric device <NUM>, and/or a housing <NUM>. In many embodiments, the hemispherical prism <NUM>, the high NA lens <NUM>, and the housing <NUM> can be similar to the aforementioned hemispherical prism (<NUM> (<FIG>) or <NUM> (<FIG>)), the high NA lens (<NUM> (<FIG>) or <NUM> (<FIG>)), and/or the housing (<NUM> (<FIG>) or <NUM> (<FIG>)), respectively.

In many embodiments, the thermoelectric device <NUM> can be configured to control the temperature of the SPR sensor <NUM>, such as maintaining the temperature fluctuation of the SPR sensor <NUM> within <NUM> degrees Celsius (°C), in order to avoid noises such as baseline shift in the SPR response signal or change in SPR angle. In some embodiments, the thermoelectric device <NUM> can be located within the optical assembly <NUM>, such as near the top surface of the hemispherical prism <NUM>, and/or partially or entirely surrounding the hemispherical prism <NUM>, in order to maintain the temperature of the hemispherical prism <NUM> and, in turn, maintain the temperature of the SPR sensor <NUM>.

In many embodiments, the microfluidic device <NUM> can be mountable on the SPR sensor <NUM> and configured to deliver a buffer solution with one or more ligand samples <NUM> onto the metal-coated sample contacting surface of the SPR sensor <NUM>. In some embodiments, the microfluidic device <NUM> also can comprise: a pump with buffer exchange and degas capability configured to control a flow of the buffer solution; and/or an auto-sampler configured to place one or more ligand samples onto the metal-coated sample contacting surface of the SPR sensor <NUM>.

In many embodiments, the SPR imaging system <NUM> can comprise the sensor translation mount <NUM> to monitor more cell population on a single SPR sensor <NUM>. In many embodiments, the sensor translation mount <NUM> can be mountable on a planar top surface <NUM> of the hemispherical prism <NUM> and configured to hold the SPR sensor <NUM> and translate or move the SPR sensor <NUM>, by an x adjust arm <NUM> (<FIG>) and a y adjust arm <NUM> (<FIG>), on the planar top surface <NUM> of hemispherical prism <NUM>, e.g., within a few millimeters on the planar top surface <NUM>, to expand the measuring area on the SPR sensor <NUM>, such as a measuring area as much as <NUM> times the area of the optical field of view of the SPR sensor <NUM>. In many embodiments, a thin layer of matching oil (not shown) also can be applied between the SPR sensor <NUM> and the planar top surface <NUM> of the hemispherical prism <NUM> to eliminate any interface effect and provide lubricant for sensor translation. In some embodiments, the top surface of the hemispherical prism <NUM> is not planar or is not entirely planar.

Turning to the drawings, <FIG> show an SPR imaging system <NUM>, according to an embodiment. In many embodiments, the SPR imaging system <NUM> can comprise a motorized frame <NUM> configured to change an incident angle ø of a low-coherent monochromatic light beam <NUM> relative to an optical assembly <NUM>. As known in the art, a typical intensity-angle profile curve comprises a dip at the SPR angle, and an angle spread between the two steep slope regions near the dip is where a small angle shift can cause significant change in an intensity of SPR light shown in an SPR image; thus, measuring at the angle spread provides the highest sensitivity for SPR measurement. In many embodiments, the SPR imaging system <NUM> can be configured to automatically scan and/or adjust the angle ø of an incident low-coherent monochromatic light beam <NUM> to provide a wide range of angle adjustment, e.g., <NUM> - <NUM> degrees. The angle adjustment allows the detector to optimize the sensitivity in SPR measurement. For instance, in some embodiments, the motorized frame <NUM> configured can be configured to move the SPR light source <NUM> vertically or in one linear direction only to adjust the light path and then the incident angle ø of the low-coherent monochromatic light beam <NUM>.

Additionally, as known in the art, the stability of an SPR light source, such as an SPR light source <NUM>, can have a significant effect on SPR response signal measurement, and the measured reflectivity intensity Im is proportional to the incident light intensity Io times a function a(n) of sensor surface property and refractive index at and/or near its surface. That is, Im = a(n) x Io. To minimize this dependency of Io, a normalized SPR signal Ispr can be used:
Ispr = Im / Io = a(n), independent of Io.

Therefore, in many embodiments, the SPR imaging system <NUM> can further comprise a light intensity detector <NUM> (<FIG>) configured to monitor and record the intensity of the low-coherent monochromatic light beam <NUM> for intensity normalization and noise reduction. In these and other embodiments, the SPR imaging system <NUM> also can comprise a beam splitter <NUM> (<FIG>), such as a <NUM>% beam splitter, located in the light path of incident low-coherent monochromatic light beam <NUM> to split the incident low-coherent monochromatic light beam <NUM> into two portions. In many embodiments, with the light intensity detector <NUM> and/or the beam splitter <NUM>, the SPR imaging system <NUM> can be configured to automatically detect and/or maintain the intensity of the low-coherent monochromatic light beam <NUM>.

In an embodiment, a system can comprise an optical assembly, an SPR light source, and/or an SPR camera. The optical assembly in this and other embodiments can comprise a hemispherical prism that comprises a top surface configured to support a surface-plasmon-resonance (SPR) sensor; a high numerical aperture (NA) lens; and a housing configured to mount the hemispherical prism and the high NA lens such that the high NA lens is located distal from the top surface of the hemispherical prism. The SPR light source in this and other embodiments can be configured to emit a low-coherent monochromatic light beam for SPR imaging toward the high NA lens. The SPR camera in this and other embodiments can be configured to capture an SPR image formed after the low-coherent monochromatic light beam is incident upon and reflected by a metal-coated sample contacting surface of the SPR sensor. Additionally, in this and other embodiments, the high NA lens can be configured to refract the low-coherent monochromatic light beam from the SPR light source toward the hemispherical prism; and the hemispherical prism can be configured to collimate the low-coherent monochromatic light beam, as refracted by the high NA lens, toward the SPR sensor.

In another embodiment, a method for surface-plasmon-resonance (SPR) imaging can comprise: (a) coupling an SPR sensor to an optical assembly; (b) placing one or more ligand samples on a metal-coated sample contacting surface of the SPR sensor; (c) emitting a low-coherent monochromatic light beam from an SPR light source toward the high NA lens; and/or (d) capturing an SPR image by an SPR camera. In this and other embodiments, the optical assembly can comprise a hemispherical prism comprising a top surface configured to support the SPR sensor; a high numerical aperture (NA) lens; and/or a housing configured to mount the hemispherical prism and the high NA lens such that the high NA lens is located distal from the top surface of the hemispherical prism. In addition, in many embodiments, the high NA lens can be configured to refract the low-coherent monochromatic light beam from the SPR light source toward the hemispherical prism. Further, the hemispherical prism in these and other embodiments can be configured to collimate the low-coherent monochromatic light beam, as refracted by the high NA lens, toward the SPR sensor. Moreover, in these and other embodiments, the SPR image can be formed after the low-coherent monochromatic light beam is incident upon and reflected by the metal-coated sample contacting surface of the SPR sensor.

Claim 1:
A system (<NUM>) comprising:
an optical assembly (<NUM>) comprising:
a hemispherical prism (<NUM>) comprising a top surface (<NUM>) configured to support a surface-plasmon-resonance (SPR) sensor (<NUM>); and
a high numerical aperture (NA) lens (<NUM>) located distal from the top surface (<NUM>) of the hemispherical prism (<NUM>);
an SPR light source (<NUM>) configured to emit a light beam (<NUM>) for SPR imaging; and
an SPR camera (<NUM>) configured to capture an SPR image; and
a bright field light source (<NUM>) and a bright field camera (<NUM>) for bright field imaging;
wherein:
the high NA lens (<NUM>) is configured to refract the light beam (<NUM>) toward the hemispherical prism (<NUM>);
the hemispherical prism (<NUM>) is configured to collimate the light beam (<NUM>), as refracted by the high NA lens (<NUM>), toward the SPR sensor (<NUM>); and
the high NA lens (<NUM>) is further configured to receive and refract the light beam (<NUM>) toward the SPR camera (<NUM>), after the light beam (<NUM>) is reflected by a surface of the SPR sensor (<NUM>);
the bright field light source (<NUM>) is configured to emit a bright field light beam (<NUM>) to illuminate the surface of the SPR sensor (<NUM>); and
the bright field camera (<NUM>) is configured to capture a bright field image of the SPR sensor (<NUM>) simultaneously with the SPR camera (<NUM>) capturing the SPR image.