This invention generally relates to optical imaging and, more particularly, to a miniaturized optical imaging system suitable for Surface Plasmon Resonance imaging.
In Surface Plasmon Resonance (SPR) imaging, an optical imaging system is used to observe biomolecular binding events that have spatial structure. Generally, such a system includes a light source to illuminate a sensor substrate under conditions which produce SPR and a detector to image the light reflected from the sensor substrate. As known in the SPR art, the sensor substrate is typically provided with a resonance layer of metal, such as gold (e.g., 50 nm thick layer). The side of the gold layer opposite from the sensor substrate faces an aqueous sample (including biomolecules), which is typically contained in a flow cell (a detector sensor volume). Light is directed through the sensor substrate and the gold layer to the aqueous sample, and is reflected therefrom. The amount of light reflection varies depending on the change in the refractive index (RI) of the sensor substrate upon adsorption of target biomolecules to the sensor substrate.
Specifically, for certain wavelengths and angles of incident illumination, part of the incident energy couples into a surface plasma wave traveling between the gold layer and the aqueous sample. The loss of this energy is observed as a decrease in reflectivity. Because the coupling conditions vary widely with the refractive index of biomolecules, observations of reflectivity may be used as a sensitive measure of the sample's refractive index, and hence of the biomolecules contained in the sample. Therefore, SPR reflectivity measurements can be used to detect various target biomolecules, such as proteins. To make an SPR imaging system for detection of specific biomolecules, the side of the gold layer, with which the sample interfaces, may be chemically functionalized (for instance, by attaching antibodies to the surface), such that the target biomolecules will bind to the sensor substrate while other material will tend not to bind. If the functionalized layer on the gold layer is patterned such that different regions tend to bind different biomolecules, the changes in reflectivity may be analyzed to determine which of a number of different biomolecules are present in the (aqueous) sample, and in what concentration. In other words, in SPR imaging, any spatial variation in surface RI across the sensor substrate causes the reflected intensity to have spatial structure. SPR imaging can be used, for instance, in medical diagnostics, to analyze a fluid (such as blood or saliva) and determine the concentration of a certain set of biomolecules in that fluid.
Referring back to
Referring additionally to
The present invention extends the SPR imaging system described above, to provide a miniaturized and highly-portable SPR imaging system having particular applicability to medical diagnostics and life sciences research and development.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
In accordance with various exemplary embodiments of the present invention, a miniaturized Surface Plasmon Resonance (SPR) imaging system is provided, which includes a light source, a sensor substrate arranged to receive light at an incident angle from the light source, and a detector for detecting an image from the sensor substrate. The system further includes a folded light path structure arranged between the light source and the detector. The folded light path structure includes the sensor substrate, and is configured so as to receive the light from the light source, to redirect the received light to be incident on the sensor substrate at the incident angle (first redirection), and to further redirect the light reflected from the sensor substrate toward the detector (second redirection). The folded light path structure achieves a minimal instrumentation footprint, which in turn allows for the construction of a miniaturized, hand-portable SPR imaging system.
In accordance with one aspect of the invention, the miniaturized SPR imaging system further includes at least one input optical element between the light source and the folded light path structure, such as one or more of a collimation lens, wavelength-selection filter, and a polarizer. When a polarizer is included, the system may be advantageously configured to obtain a TE-polarized reference image for normalizing a TM-polarized image of the sensor substrate. The system may still further include at least one output optical element between the light path structure and the detector, which is capable of accepting light reflected from the sensor substrate. In one embodiment of the invention, the output optical element may include a pair of biconvex lenses and an achromatic lens. In accordance with a further aspect of the invention, wherein the output optical element comprises a lens, the light source, the detector, and the lens are arranged relative to each other so as to satisfy the Scheimpflug condition, thereby achieving sharp focus of the image of the sensor substrate.
In accordance with another aspect of the invention, the miniaturized SPR imaging system is further configured to be capable of adjusting focus and/or magnification of an image of the sensor substrate.
In accordance with a further aspect of the invention, the folded light path structure is configured such that the optical path resulting from the first redirection and the second redirection lies approximately in parallel with the sensor substrate. Also, the folded light path structure may include two symmetrical substructures on the light source side and the detector side, respectively. In one embodiment of the invention, such folded light path structure is formed with one main prism, one redirection optical element provided on each of the two sides of the main prism, and one deflection optical element also provided on each of the two sides of the main prism.
In accordance with yet another aspect of the invention, the folded light path structure includes one or more optical elements and the folded light path structure is further configured to adjust the one or more optical elements relative to the light source and the detector. For example, the one or more optical elements are adjustable at least linearly along one direction and angularly along the optical axis of the miniaturized SPR imaging system. Likewise, the light source and the detector may be adjustable at least linearly along one direction and angularly along the optical axis of the miniaturized SPR imaging system.
In accordance with a still further aspect of the invention, the incident angle of the light from the light source on the sensor substrate is adjustable. This could be accomplished, for example, by configuring the light source to selectively emit illumination light from two or more locations along a line perpendicular to the optical axis of the miniaturized SPR imaging system.
In accordance with further embodiments of the present invention, a miniaturized Surface Plasmon Resonance (SPR) imaging instrument is provided, including a miniaturized SPR imaging system described above. The SPR imaging instrument further includes a case that houses the miniaturized SPR imaging system, such that the sensor substrate is exposed to the outside of the case. The case also houses an electronic board for controlling the operation of the SPR imaging instrument. The SPR imaging instrument additionally includes a microfluidic card configured to be positioned adjacent to the exposed sensor substrate so as to subject an aqueous sample flowing therethrough to SPR imaging. Finally, the SPR imaging instrument includes a computer including a user interface to guide a user through the operation of the SPR imaging instrument. The computer is further loaded with suitable image processing and analysis software.
In accordance with one aspect of the invention, the microfluidic card comprises a window layer, a resonant (gold) layer formed on the window layer, and a flow layer formed on the resonant layer and including fluid paths for flowing the aqueous sample therethrough. The microfluidic card may be further configured to be mated with external fluidics that input and output the aqueous sample to be analyzed.
In accordance with another aspect of the invention, the miniaturized SPR imaging instrument further includes means for stabilizing the internal temperature of the case, such as a fan and/or a heater, to reduce and minimize wavelength drift or changes in a polarizer.
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
In accordance with various embodiments, the present invention offers a miniaturized optical system with a small instrumentation footprint, particularly designed to allow a highly sensitive SPR (SPR) bio-assay to be conducted over a sensor substrate that can contain many different simultaneous assays. The system utilizes an optimized imaging technique to resolve the assay response for very small localized regions anywhere in its field of view. The optical path through the system is designed to be very rugged and compact, with few or no moving parts. There are provisions to allow the response of the system to be tuned to optimize the sensitivity of the assay for a given protocol chemistry or for a particular set of samples.
In the illustrated embodiment, light rays (three are shown) 29 emitted from the light source propagate through the illumination optics 22 and strike the hypotenuse plane of the 90°-deflection prism 24a, from which the rays 29 are reflected to enter the first redirection equilateral prism 25a to strike its plane 25b, from which the rays 29 are reflected to enter the main prism 26a through one of its side planes 26b. The rays 29 then strike a sensor substrate 26c of the main prism 26a, and are totally internal-reflected therefrom to exit the main prism 26a through the other of its side planes 26d, to enter the second redirection equilateral prism 27a to strike its plane 27b. The rays 29 reflected from the plane 27b exit the second redirection equilateral prism 27a to enter the second 90°-deflection prism 28a to strike its hypotenuse plane 28b, from which the rays 29 are reflected to exit the second 90°-deflection prism 28a to enter imaging optics 30, such as lens(es), to be finally received by an image detector (not shown) on a detector mount 31. Thus, an image of the sensor substrate 26c is formed on the detector. Note that the sensor substrate 26c of (or on) the main prism is provided with a metal layer, such as gold, on the side that faces an aqueous sample to be analyzed (i.e., the top side in
As should be apparent to those skilled in the art, the folded light structure 23 illustrated in
In the illustrated embodiment of
The following describes in detail various subsystems of a miniaturized SPR imaging system formed in accordance with various exemplary embodiments of the present invention. Thereafter, implementation of a complete miniaturized SPR imaging instrument will be described.
Illumination Subsystem
As used herein, the illumination subsystem includes the light source 42, the illumination optics 22, and the folded light path structure 23. The signal produced by an SPR imaging system is highly dependent on the wave vector of the illumination. Therefore, preferably, the illumination subsystem illuminates the entire sensor substrate 26c with light that has a uniform wave vector and radiance. Achieving uniform radiance helps optimize the signal-to-noise ratio (SNR) in the imaging system. Also, a non-uniform wave vector could result in different portions of the sensor substrate 26c having different response calibration, yielding complicated nonlinear results. Different assay chemistries require different wave vectors of illumination for optimal response. Therefore, it is further preferable that the illumination subsystem allows the illumination wave vector to be adjusted to achieve optimized sensitivity.
One way to adjust the wave vector is to fix the spectral content of the illumination and then vary angle of the illumination on the sensor substrate 26c. In one embodiment of the present invention, this is achieved by using a light source 42 with a narrow spectral width and moving the light source 42 relative to the illumination (collimation) optics 22. The illumination (collimation) optics 22 are chosen to produce a beam with a uniform wave vector for a range of light source positions relative to the optical axis of the imaging system. When the light source 42 is translated relative to the optical axis, as shown in
Another way to adjust the wave vector is to fix the angular content of the beam and vary the wavelength content. In one embodiment of the present invention, this is achieved by putting a broad wavelength source through an interference filter to narrow the spectral width of the output. Such interference filter may be provided as part of the illumination optics 22. If the filter is tipped, the central wavelength of the output beam can be varied over a range of wavelengths. In this fashion, the output beam will still have a uniform wave vector across its width, but the wave vector will vary with wavelength. This spectral adjustment can also be accomplished using techniques such as, without limitation, rotating prisms or diffractions gratings, but these may not produce a very uniform wave vector across the beam. Using a technique such as, without limitation, a fiber optic to mix the output prior to collimation will help solve this issue.
The light source 42 may comprise, without limitation, one or more LEDs with narrow spectral width at 660 nm, 880 nm, or 920 nm; one or more LEDs with a broad spectral width, an incandescent source, a fluorescent source, a laser source, an optical fiber, among others, either alone as a single point source, as a line source (such as a row of LEDs or a rectangular fiber bundle, thereby improving flux with increased light throughput without significantly increasing the range of wave vectors in the illumination beam), or as a two-dimensional array.
The techniques for adjusting the light source 42 may include, without limitation, a single light source provided on a moving mount that is driven by a manual or motorized actuator, an array of light sources where each source can be individually controlled such that only the source(s) that produces the optimal wave vector is turned on, and a wavelength-selection filter (e.g., as part of the illumination optics 22) on a moving mount driven by an actuator, which causes the filter to tip relative to the optical axis.
Polarization Control
The signal produced by an SPR imaging system is highly dependent on the polarization state of the illumination beam. When the beam is TE (Transverse Electric) polarized relative to the sensor substrate 26c, no SPR occurs and the image recorded by the detector 60 will represent the illumination transfer function of the entire imaging system. When the illumination beam is TM (Transverse Magnetic) polarized, an SPR imaging signal can be recorded by the detector 60. However, this signal will also contain all the non-uniformities caused by the rest of the system throughput. In accordance with various exemplary embodiments of the present invention, the TE reference image is used as a “real time” reference to normalize the TM image so as to remove the non-uniformities, leaving only the SPR image signal from the sensor substrate 26c. In one embodiment of the present invention, a liquid crystal element (as part of the illumination optics 22) is used to rotate the polarization state of the incident illumination by applying a voltage to the liquid crystal element. Other techniques for varying the polarization state include, without limitation, an actuator-driven stage to rotate a piece of linear polarizer.
Folded Light Path
In various exemplary embodiments of the present invention, the optical paths of the illumination and SPR imaging signal light are folded up in an approximately symmetrical fashion using identical components, as described in reference to
In this embodiment, the illumination beam is first folded by the first 90°-deflection prism 24a with its mirrored hypotenuse plane, to be perpendicular to the sensor substrate 26c. While the prism 24a is used in this embodiment to simplify the mounting and cleaning of optics, the 90°-deflection may also be achieved with a mirror. Whatever deflection optic is used, it may be mounted on (or in) an adjustable mount that allows three axes of angular adjustment and one axis of linear adjustment. For example, referring additionally to
After the deflection optic, the beam is redirected from the normal (i.e., perpendicular to the sensor substrate 26c) to the sensor substrate 26c so that it illuminates the backside of the sensor substrate 26c at a grazing angle. In the embodiment illustrated in
One advantage of using a redirection prism 25a is that it can be bonded to the main prism 26a. This provides a simple way to construct the miniaturized SPR imaging system 20. In other embodiments, the main prism 26a may be constructed with a monolithic shape that has the characteristics of a redirection prism bonded to a main prism.
In the embodiment illustrated in
Mounting the Main Prism and Sensor Substrate
In one embodiment of the present invention, the main prism 26a is bonded to one side of a large, flat glass window 32, the other side of which is coated with gold. The sensor substrate 26c is index-matched to the window 32 so that the center of the sensor substrate 26c intersects the optical nominal axis of the illumination beam. The use of the window 32 is advantageous in that it provides a simple way of holding the main prism 26a in proper position. Further, the window 32 is easy to mount to the rest of the SPR imaging system 20, and also serves as a flat support surface to ensure that the sensing surface (as provided by the gold layer) remains flat. Finally, the window 32 helps protect the rest of the SPR imaging system 20 by minimizing any sample leaks and also containing excessive amounts of index matching.
Imaging Optics and Detector
The imaging optics 30 are responsible for creating a crisp image of the sensor substrate 26c on the detector 60. Because the sensor substrate 26c is tipped relative to the optical axis of the illumination side, the detector 60 also should be tipped in a configuration known as the Scheimpflug condition to achieve best focus, as discussed above. In accordance with various exemplary embodiments of the present invention, it may be further preferable to magnify the image by a factor of 1.5, a condition that makes the SPR imaging system more linear than can be achieved by simply obeying the Scheimpflug condition. It is still further preferable that the image remains stable on the detector 60 even when the angle of the illumination beam is adjusted.
To achieve one or more of these preferable features, in one embodiment of the present invention as illustrated in
In one embodiment of the present invention, the detector 60 is a rectangular (e.g., 4.8 mm×3.6 mm) CCD array detector placed at the image plane 58 of the SPR imaging system. In various exemplary embodiments of the present invention, the detector 60 is mounted in such a way that it can be translated in three axes and rotated in two axes in order to achieve optimal focus. Preferably, the detector 60 is at least rotatable around the optical axis in order to account for any tip and tilt imparted by the mounts of the deflection prisms (e.g., the first and second 90°-deflection prisms 24a and 28a). At the same time, the illumination subsystem (comprising the light source 42, the illumination optics 22, and the folded light path structure 23) is preferably rotatable around its optical axis to keep everything aligned.
Stable Thermal Environment
To achieve a stable SPR imaging signal, it is preferable that the thermal environment for the optical path either remains constant or is corrected for by the software. Software correction typically works only over a relatively small temperature range, so a sound thermal design is preferable for a system to be stable over a relatively large temperature range. Temperature variation can cause the wave front to wander due to: mechanical variation in the mounting structure, wavelength drift of the light source 42, wavelength drift of any optical filters (if used as the light source 42), and/or changes to the polarizer.
In one embodiment of the present invention, the temperature is stabilized slightly above ambient temperature using a fan to circulate the air inside a case (containing the optics) and a heater on a controller to add just enough heat. Another embodiment uses a circulating fan and a heat pipe to pull the heat from the imaging electronics out of the case. An external fan on a controller may also be used to control the amount of heat conducted out of the case. Those skilled in the art will appreciate that various other techniques to stabilize the internal temperature of the system are also within the scope of the present invention.
Hyperspectral Imaging
The embodiments discussed so far are intended to monitor an illumination beam with a stable wave vector over a period of time to establish an assay for a given location. This is a single point measurement scheme, and even with careful normalization against a “real time” reference image (e.g., the TE reference image) and careful stabilization of the system, it is prone to undesirable wavelength drift. This drift can be minimized or eliminated by moving from a single point measurement to a multipoint measurement. One way to accomplish this is to measure a range of wave vectors at each location in rapid succession. It is then possible to recover a more stable signal using signal processing techniques such as, without limitation, ratiometric techniques, minima hunting, correlation techniques, or regression techniques.
One technique of achieving such data from a range of wave vectors is to time share the detector 60. If the illumination beam is fixed at an angle and a series of successive images are taken at different wavelengths, the spectra for each pixel on the detector 60 can be built up from the same corresponding pixel in the series of images. This is a technique referred to as hyperspectral imaging. A similar set of hyperspectral data can also be taken with a fixed wavelength and a rapid series of images at different illumination angles. In various exemplary embodiments of the present invention, the use of hyperspectral image processing improves the functionality of the SPR imaging system.
Implementation of the Complete Instrument
The miniaturized SPR imaging system with a small footprint, as described above, may be readily incorporated into a complete SPR imaging instrument 70, as shown in
(a) Microfluidic Card
Referring additionally to
(b) DSP Software
In one embodiment of the present application, a Texas Instruments TMS320C6410 DSP may be used to control the CCD image detector 60 that captures the reflected light and to transfer the resultant SPR images to the computer 76 (e.g., the tablet PC) for processing and analysis. The specific functions of the DSP software, which was developed in the Texas Instruments Code Composer Studio software development environment, include setting the image detector shutter speed, acquisition of images from the detector, averaging the images to remove temporal noise, and transmission of the averaged images via Ethernet to the tablet PC.
(c) Tablet PC Software
The computer (e.g., tablet PC) software is responsible for control of the SPR imaging instrumentation, processing and analysis of the averaged SPR images, and for providing a simple user interface to guide a user through the experimental procedure. In one embodiment, the instrumentation control and user interface software were developed in the National Instruments LabWindows/CVI software development environment, while the SPR image processing and analysis software was developed in the Mathworks Matlab environment.
In one embodiment of the present invention, the image acquisition software module connects to the DSP via two Ethernet channels. The first channel is used to launch the program on the DSP. The second channel is used for transmitting images from the DSP and sending to the DSP the desired image detector shutter speed and number of images for averaging. Each image received from the DSP may be stamped with the time it was received from the DSP, the number of images averaged, and the shutter speed, before being saved to disk for processing and analysis. In addition, the most recently acquired image may be displayed on the computer 76 screen at a reduced resolution.
The flow of fluids through the SPR imaging instrument 70 may be controlled by a set of six Kloehn syringe pumps, which is part of the external fluidics 72. In one embodiment, each pump, which contains a 6-way valve, syringe, and stepper motor, is individually programmed via an RS-232 serial connection. The software sets the valve position and determines the distance and velocity for syringe activity in units of motor steps based on the syringe volume, fluid flow duration, fluid flow direction (aspirate or dispense), and fluid volume.
The intensity of the light source 42, such as an LED light source, may be set via a codeword corresponding to the current to be sent to the LEDs. The codeword is transferred to the electronic control board via a serial connection using the RS-232 protocol.
The rotation angle for the electronic polarizer (as part of the illumination optics 22) may be set via a codeword transferred to the electronic control board via an RS-232 serial connection. In one embodiment, a codeword of zero corresponds to TM polarization and a codeword of 1974 corresponds to TE polarization.
In one embodiment, a set of four valves (part of the external fluidics 72) controls the flow path of fluids in the microfluidic card 74b. The software sets each valve to either an open or closed state using a binary code, which is transferred to the electronic control board via an RS-232 serial connection. In one embodiment, three of the valves operate in unison, thus requiring a two-bit code.
(d) User Interface
In accordance with various exemplary embodiments of the present invention, the SPR imaging instrument 70 offers a graphical user interface consisting of multiple panels, to guide users through the experimental procedure. The main panel 91 of the user interface, which is shown in
In the illustrated embodiment, five of the buttons 92 correspond to a fixed set of pump and valve operations, thereby eliminating the need for the user to be aware of fluid flow rates and volumes. The sixth button 94 adjusts the polarizer and light source for the acquisition of dark, TE-polarization, and TM-polarization reference images. Finally, a “STOP” button is available to immediately terminate all pump activity.
For advanced control of the SPR imaging instrument 70, as shown in
(e) Image Processing and Analysis
As discussed above, one embodiment of the image processing and analysis software was implemented in Mathworks Matlab, to provide on-the-fly experiment results for the user. The user interface for the image processing and analysis software is shown in
The initialization procedure automatically finds the directory containing the current set of SPR images, initializes the regions of interest (e.g., rectangular areas used for data analysis) in the images, loads the dark, TE and TM reference images, and sets up the graphs for the data display.
In one embodiment, the analysis routine, which checks for images periodically based on a timer, gives the user a choice of four ways to visualize data. The first method is a plot of the mean spatial intensity of each region of interest over time. The user also has the option of specifying whether the images are corrected for background, dark, and illumination noise. Second, the images can be subtracted from a reference image to show differences in intensity from a known point in time. Two other ways to visualize the data are via horizontal and vertical line profiles. Third, specifically, a single row or column of each image is displayed as a line plot depicting intensity as a function of spatial location. The same line from each progressive image is appended to the display using a different color to allow temporal analysis of the line. Alternatively, multiple rows or columns can be averaged together for the line profiles. The fourth data visualization method is a histogram of pixel intensities that is destructively updated for each image.
As described hereinabove, the present invention, in accordance with various exemplary embodiments, offers a miniaturized SPR imaging system with a minimal footprint, which in turn makes possible a compact and portable SPR instrument containing such miniaturized SPR imaging system. In addition to being compact, the miniaturized SPR imaging system of the present invention includes a robust optical path that may be adjustable (in terms of focus, magnification, illumination wavelength, illumination incident angle, etc.) so as to achieve optimal illumination onto the sensor substrate and optimal imaging onto the detector. The miniaturized SPR imaging system is also designed to be mechanically robust in order to hold its adjustment. Further features may be provided to allow the image to be stable for a range of conditions of the light source illumination, including changes in the wavelength, angle of incidence, and polarization.
The SPR instrument in accordance with the present invention enables rapid, convenient desktop monitoring of biochemical binding interactions, for instance for immunoassays. The miniaturized SPR imaging system described herein is useful for many applications, including those requiring (1) detection and/or quantification of biological binding events; (2) detection and/or quantification of other binding or adsorption processes; or (3) refractometry of substances or surfaces which have a spatial distribution. Thus, it has particular applicability to medical diagnostics and life sciences research and development. While this invention is generally directed to a miniaturized high-performance SPR imaging system, it will be appreciated by those skilled in the art that the optical imaging system disclosed herein could be useful for many applications. Other angle-dependent optical sensing techniques such as ellipsometry and Brewster angle microscopy will likewise benefit, as will imaging or illumination systems in which facile adjustment of illumination conditions is needed.
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
This application claims the benefit of U.S. Provisional Application No. 60/814,253, filed Jun. 16, 2006, which is hereby expressly incorporated by reference.
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of 1 U01 DE14971-03 awarded by the National Institutes of Health.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US07/71471 | 6/18/2007 | WO | 00 | 1/28/2010 |
Number | Date | Country | |
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60814253 | Jun 2006 | US |