WO2010121165A1

WO2010121165A1 - Method and apparatus for optical interferometric measurements using a reference region

Info

Publication number: WO2010121165A1
Application number: PCT/US2010/031436
Authority: WO
Inventors: David Alan Bergstein
Original assignee: Zoiray Technologies Inc.
Priority date: 2009-04-16
Filing date: 2010-04-16
Publication date: 2010-10-21

Abstract

The invention features planar substrates and methods for optical interferometric measurements. Provided herein are planar substrates that include one or more reference regions and methods of making such planar substrates. Also provided are methods for determining an interference signal that is normalized to account for noise including the detecting an interference signal from one or more reference regions.

Description

PATENT

ATTORNEY DOCKET NO. 50710/003WO2

METHOD AND APPARATUS FOR OPTICAL INTERFEROMETRIC MEASUREMENTS USING A REFERENCE REGION

Background of the Invention The invention relates to optical interferometric measurements of substrates.

It is expected to be particularly useful for detecting molecular binding interactions in an array format on a substrate label-free by interference measurement.

Optical interference ha?; been employed to monitor height or mass changes on a surface or in a thin layer. For examples, see the works of Saadstrδm et al, (Appl. Opt. 24:472-479, 1985), Jenison et al. {Nat. BiotechnoL 19:62-65, 2001 ), Gauglitz {Rev. ScL Instrum. 76:062224, 2005), Nikitin et al. {Sensor. Actual B 111-112:500-504, 2005), Bergstein et al. {IEEE J. SeI. Top Quantum Electron. 14:131-139, 2008), and Ozkumur et al. {Proc. Natl Acad, Set USA 105:7988- 7992, 2008). Note in particular that Nikitin' s picoscope, Gauglitz 's Reflectometric Interference Spectroscopy (RIfS), Bergstein's Resonant Cavity Imaging Biosensor (RCIB), and Ozkumur' s Spectral Reflectance Imaging Biosensor (SRIB) all measure molecular binding events across a surface by optical interference, or in the case of RCIB, principally resonance. The interference signals in the case of the picoscope, RIfS, and SRIB are the result of optical resonance within a semitransparent thin layer. The optical principles of thin film interference are well described by Yeh {Optical Waves in Layered Media New York: John Wiley & Sons, 1988, pp. 416). RCIB is different in that the signal results from a resonant structure with multiple high finesse resonant cavities coupled together, while picoscope, RIfS, and SRIB all use low finesse optical cavities. Despite their differences, optical interference from multiple reflecting boundaries is the principle mechanism for picoscope, RIfS, SRIB, and RCIB. Together these will be classified along with other techniques that invoke PATENT

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interference of reflections from multiple boundaries to detect changes on a surface or within a layer as "optical interference measurements."

A key application of optical interference measurement is the imaging of bioarrays, such as DNA microarrays or protein arrays, as described by Stears et al. {Nat. Med. 9: 140- 145, 2003) for both biomedical research and medical diagnostics. These and other techniques that invoke interference of reflections from multiple boundaries to detect changes on a surface or within a layer and do so across a surface at a plurality of locations in parallel are referred to herein as "imaging optical interference measurements" (IOIM), Whenever one performs IOIM, it is critically important that the intensity of the incident light be maintained constant or otherwise monitored and normalized later in signal processing. Light sources, whether laser, lamp, LED, or other, are subject to possible deviations or fluctuations in light output. Causes for intensity fluctuations of the source include temperature changes due to heating, electric power fluctuations, or many other random or predictable events. These fluctuations create an additional signal on the interference measurement that introduces noise into the measurement and reduces the measurement accuracy. It is desirable then, for high accuracy and high precision measurements, to compensate for this effect. One common method is to monitor the light level by use of a photodiode and to use the monitored intensity as feedback to maintain the light intensity. Many sources have such power stabilization included and integrated with lamps, lasers, and LEDs, though it may also be constructed and implemented by the user. Alternatively, one might record the light intensity with a photodetector during measurement and subsequently compensate for fluctuations during the measurement in post-processing on a computer. We will refer to the first case as ''closed loop feedback on the source" and the second as ^Ckopcn loop post-processing compensation." PATENT

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Both of the above- described methods are used in many optical systems, and each is problematic. In the case of closed loop feedback on the source, there is generally a time constant to the feedback such that it is not instantaneous. Furthermore, there is also generally an error, such that the fluctuations are not fully compensated. In the case of open loop post-processing compensation with a photodetector, there is generally a problem that the photodetector measurement and the interference measurement (typically by a camera) are not coincident. A second problem is that the light directed to the light monitor (typically a Si photodiode) and the interference monitor (typically a digital camera or CCD) need not yield the same signal, especially for all wavelengths and possibly due to differences in polarization somewhere along the differential path. While a wavelength dependency may also be characterized, this raises an additional concern that the wavelengths used do not shift or change since the dependency was last evaluated. Additionally, evaluating such a dependency is not trivial without an accurate wavemeter included in the system. Moreover, the characteristics of the photodiode and its response to light may not be well matched to the characteristics of the camera in the case that a photodiode is used to monitor the light intensity and a camera is used to monitor the interference.

Another critical problem with closed loop feedback on the source and open loop post-processing compensation is that the amount of light recorded by a photodiode monitor and the camera is generally dependent on the polarization of the light for at least part of the path that is uncommon to these detectors. Polarization of the light can determine both how much light reaches either monitoring device and how much light either device records. The polarization may be changing in time for various sources and effect the ability of these methods to compensate for intensity fluctuations.

Most generally, separate detectors are plagued by any noise source that affects them differently, so called "differential noise,^'5 One would prefer so called PATENT

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"common mode noise" that would affect both similarly such that it can be characterized and compensated. Other differential noise sources include vibrations that shift optical components in time differently in either beam path, changes in polarization, changes in wavelength dependence, changes in intensity coupling, changes in measurement timing, and changes in readout electronics and signal quantification.

Summary of the Invention

The invention provides planar substrates and methods that provide improved measurements of optical interference. The invention uses one or more reference regions that allow for correction of inaccuracies caused by noise.

Accordingly, the invention features a planar substrate having a top face and a bottom face, the planar substrate including: a first specular reflecting interface at the top face of the planar substrate; a second specular reflecting interface that is substantially parallel to and underlies the first specular reflecting interface; one or more reference regions disposed on or within the first specular reflecting interface; and one or more measurement regions disposed on the first specular reflecting interface.

In one embodiment, the planar substrate further includes: a first layer having a top face and a bottom face, wherein the first specular reflecting interface is at the top face of the first layer; and a second layer having a top face and a bottom face, wherein the top face of the second layer is connected to the bottom face of the first layer and the second specular reflecting interface lies between the first layer and the second layer. In another embodiment, the planar substrate further includes a first layer having a top face and a bottom face, wherein the first specular reflecting interface is at the top face of the first layer and the second specular reflecting interface is at the bottom face of the first layer. PATENT

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In some embodiments, the first layer includes one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide. In other embodiments, the second layer includes one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.

In particular embodiments, at least one reference region includes a removed portion of the first specular reflecting interface (e.g., where the removed portion exposes the second specular reflecting interface). In other particular embodiments, at least one reference region includes an added portion to the first specular interface (e.g., where the added portion includes one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxanc), poly(methyl methacrylate), or plastic).

In yet another embodiment, at least one reference region is disposed on or within the first specular reflecting interface at a distance of up to about 100 mm from at least one measurement region (e.g., about 1, 2, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 75, 80, 90, or 95 mm).

In some embodiments, at least one reference region is disposed circumferentially around at least one measurement region. In other embodiments, the measurement regions form an array and at least one reference region is disposed near or within the array.

In further embodiments, the planar substrate further includes at least one capture agent directly or indirectly bound to the top face of the planar substrate.

The invention also features a planar substrate for measuring optical interference, the planar substrate including: one or more first layers having a top face and a bottom face; one or more reference regions disposed on or within at least one first layer; and one or more measurement regions disposed on at least one first layer, wherein the one or more reference regions and the one or more measurement regions are disposed on or within at least one first layer to allow for PATENT

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simultaneously measuring the optical interference from the one or more reference regions and the one or more measurement regions.

In one embodiment, the one or more first layers include one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide.

In another embodiment, the planar substrate further includes one or more second layers having a top face and a bottom face, wherein a top face of at least one second layer is connected to a bottom face of at least one first layer. In particular embodiments, the one or more second layers include one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.

In particular embodiments, at least one reference region includes a removed portion of at least one first layer to expose an underlying second layer or to decrease a height of the at least one first layer (e.g., where 0.1-50 microns are removed).

In other embodiments, at least one reference region includes an added portion to at least one first layer (e.g., where the added portion includes one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(merhyl methacrylate), or plastic).

In some embodiments, at least one reference region is disposed on or within at least one first layer at a distance of up to about 100 mm from at least one measurement region (e.g., about 1, 2, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 75, 80, 90, or 95 mm). In other embodiments, at least one reference region is disposed circumferentially around at least one measurement region. In yet other embodiments, the measurement regions form an array and at least one reference region is disposed near or within the array. PATENT

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In further embodiments, planar substrate further includes one or more capture agents directly or indirectly bound to a top face of at least one first layer.

The invention also features a planar substrate for measuring optical interference, the planar substrate including: a plurality of first layers, each layer having a top face and a bottom face; a plurality of second layers, each having a top face and a bottom face, wherein the plurality of first layers and the plurality of second layers form an alternating stack; and at least one reference region disposed within the alternating stack. In one embodiment, at least one reference region includes a removed portion of at least one first layer to expose an underlying at least one second layer.

The invention further features a method of determining an interference signal that is normalized to account for noise, the method including: a) providing a planar substrate including one or more reference regions and one or more measurement regions; b) reflecting a light source on the planar substrate; c) detecting an interference signal from the one or more reference regions and an interference signal from the one or more measurement regions; and d) adjusting the interference signal from the one or more measurement regions as a function of the interference signal from the one or more reference regions, wherein the adjusted interference signal is normalized to account for noise. In one embodiment, at least one reference region includes a removed portion of the planar substrate (e.g., where about 0.1 microns to about 50 microns of the planar substrate is removed).

In another embodiment, at least one reference region includes an added portion to the planar substrate (e.g., where the added portion includes one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic). PATENT

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In some embodiments, the planar substrate includes one or more first layers, each having a top face and a bottom face (e.g., where one or more first layers include one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide). In other embodiments, the planar substrate further includes one or more second layers, each having a top face and a bottom face, and wherein a bottom face of at least one first layer is connected to a top face of at least one second layer (e.g., where one or more second layers include one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.). In yet other embodiments, the planar substrate includes a plurality of first layers, each having a top face and a bottom face, and a plurality of second layers, each having a top face and a bottom face, and wherein the plurality of first layers and the plurality of second layers form an alternating stack.

In particular embodiments, at least one reference region includes a removed portion of at least one first layer to expose an underlying second layer or to decrease a height of the at least one first layer (e.g., where about 0.1 microns to about 50 microns of the planar substrate is removed). In other embodiments, at least one reference region includes an added portion to at least one first layer (e.g., where the added portion includes one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic).

In some embodiments, the detecting step c) includes detecting an interference signal from a portion of the one or more reference regions.

In other embodiments, the adjusting step d) includes accounting for noise sources selected from the group consisting of intensity fluctuations of the light source, polarization effects, vibration, mechanical movement, and non-specific binding. In particular embodiments, the adjusting step d) includes adjusting as a function of a mean of the interference signal from at least one reference region. PATENT

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In some embodiments, the interference signal from at least one measurement region includes a signal from a pixel, an average signal from more than one pixel, a sum signal from than one pixel, or a mean signal from more than one pixel. In other embodiments, the interference signal from at least one reference region includes a signal from a pixel, an average signal from more than one pixel, a sum signal from than one pixel, or a mean signal from more than one pixel.

The inventions features a method of making a planar substrate including one or more reference regions and one or more measurement regions, the method including: a) masking a portion of the planar substrate to form a planar substrate having a mask, wherein the mask defines masked portions and exposed portions of the planar substrate (e.g., including applying photoresist to the planar substrate; applying a photomask to the photoresist and the planar substrate; exposing the photomask, the photoresist, and the planar substrate to a light source; and removing the photoresist that was not exposed to the light source to form the planar substrate having a mask); b) etching the planar substrate having the mask, wherein the exposed portions are etched to define at least one reference region and the masked portions define at least one measurement region (e.g., including hydrofluoric acid, buffered oxide etch, or deep reactive ion etching); and c) removing the mask, wherein the at least one reference region and the at least one measurement region are disposed on the planar substrate to allow for simultaneously measuring optical interference from the at least one reference region and at least one measurement region.

The invention also features a method of making a planar substrate including one or more reference regions, the method including adding one or more materials on the planar substrate to form at least one reference region (e.g., where the one or more materials are one or more of glass, silicon dioxide, titantium oxide, porous PATENT

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silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic).

By "analyte" is meant a molecule being detected in a sample.

By "array," when referring to measurement regions, is meant a geometric arrangement of measurement regions on a planar substrate.

By "capture agent" is meant a molecule used to detect an analyte in a sample, where the molecule is indirectly or directly bound to a planar substrate. Indirect binding includes covalent and non-covalent binding of a molecule to one or more other molecules, where these other molecules are then covalently or non- covalently bound to the planar substrate. Direct binding includes covalent and non-covalent binding of a molecule to the planar substrate. As used herein, "bound" and "binding" refers to a non-covalent or a covalent interaction that holds two molecules together or holds a molecule to a planar surface. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, electrostatic binding, van der Waals interactions, hydrophobic interactions among non-polar groups, lipophobic interactions, and LogP-based attractions.

By "interference signal" is meant a measurement of interference by an imaging optical interference measurement technique. The interference signal can be measured from any region of the planar substrate, such as the measurement region or the reference region.

By "light source" is meant any optical source used in obtaining imaging optical interference measurements.

By "measurement region" is meant a region of a planar substrate that is being measured, by any of the imaging optical interference measurement techniques described herein, to detect one or more analytes in a sample. Measurement regions can include any region that is not the reference region (e.g., the arrayed spot or areas neighboring the arrayed spot). PATENT

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By "planar substrate" is meant a surface of a slide, a chip, or a platform that can be used to obtain imaging optical interference measurements.

By "reference region" is meant a region of a planar substrate with an interference signal that is different than the resonance being measured in other regions of the planar substrate. The reference region can be any shape and cover any area of the planar substrate. The interference signal of the reference region can include no resonance or a different resonance than that observed in the measurement region.

By "specular reflecting interface" is meant an interface that reflects an incidental ray of light into a reflected ray that has a generally single outgoing direction, In some exemplary interfaces, the specular reflecting interface results in the incident ray, reflected ray, and the interface normal being coplanar.

Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.

Brief Description of the Drawings

Figure 1 is an exemplary schematic of a top view of a planar substrate (slide or chip), including reference regions (shown as gray squares), arrayed spots (or data spots, shown as black circles), and the area under test (shown as a dotted circle).

Figure 2 is another exemplary schematic of a top view of a planar substrate (slide or chip), including reference regions (shown as gray rectangles) and arrayed spots (or data spots, shown as black circles).

Figure 3 is yet another exemplary schematic of a top view of a planar substrate (slide or chip), including reference regions (shown as gray squares) and arrayed spots (or data spots, shown as black circles).

Figure 4 is an exemplary schematic of a top view of a photomask to define reference regions. PATENT

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Figure 5 is an exemplary schematic of a side view of a planar substrate (slide for SRIB), including the Si layer, the SiO₂ layer, reference regions (shown as a gap in the SiO₂ layer), and spotted antibodies for antigen capture.

Figure 6 is a schematic diagram of an exemplary SRIB system. Figure 7 is a schematic diagram of an exemplary RfIS system.

Figure 8 is a schematic diagram of an exemplary RCIB system.

Figure 9 is a schematic diagram of an exemplary experimental setup for collecting data.

Figures 1 OA and 1 OB shows an exemplary chip with an etch pattern of 20mm x 20mm. Figure 1OA is an actual CAD drawing. Figure 1OB is a schematic showing the measurement regions (area shown in white) and the reference regions (area shown in gray).

Figures 1 IA and 1 IB are CCD images of a chip. Figure 1 IA shows reference regions and the portion of the reference region used to obtain an interference signal from the reference region (shown as a black rectangle). Figure HB shows CCD images obtained at wavelengths 765 nm, 769 nm. 774 nm, and 779 nm.

Figure 12 is a graph showing a model curve for intensity versus wavelength. Figure 13 is a graph showing raw data for intensity versus wavelength from three pixels.

Figure 14 is a graph showing voltage versus wavelength for the photodetector. Figure 15 is a graph showing intensity versus wavelength for data normalized by phoiodetector voltage. Figure 16 is a graph showing intensity versus wavelength for the reference region.

Figure 17 is a graph showing mean intensity in the reference region versus photodetector voltage. PATENT

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Figure 18 is a graph showing intensity versus wavelength for data normalized by mean intensity in the reference region.

Figure 19 is a graph showing intensity versus wavelength for data normalized by mean intensity in the reference region and showing a fit to the the model curve.

Detailed Description

The present invention relates to a method and apparatus to monitor and compensate for intensity fluctuations of the light source for IOIM. According to one embodiment of the invention, a reference region is built into the substrate itself for post-processing compensation. Namely, the imaged area that produces the interference signal contains one or more reference regions that do not have the same interference signal and can be used to approximate the incident light level. In the case of SRIB, which uses an oxide on Si as the interference layer, there can be regions in which the SiO₂ is etched down to the Si layer, such that no interference will result in those regions (see, e.g., Figures 1-3).

The imaging system will collect data from both one or more reference regions (e.g., regions with no resonance or a different resonance than that observed in the measurement region) and one or more measurement regions (e.g., regions with the expected resonances shifting from binding). The reference regions can be used to determine the intensity of the incident light at the same time the interference measurement is made. It is noted that in this example, the "no resonance" regions would be insensitive to nonspecific binding since there is no resonance. It is also noted that it is not necessary to eliminate interference from the reference regions altogether, so long as the interference pattern in wavelength is appreciably different from the resonance being measured. Whether the interference pattern is spread out in spectra (e.g., in the case of SRIB, by making the oxide much thinner in the reference regions) or by compressing the PATENT

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interference pattern in spectra (e.g., in the case of SRIB, by making the oxide much thicker), it can still be used as a reference for the interference being sought since it will not be changing identically with binding.

As an example of the processing, for SRIB, the SiO₂ in the reference regions may be etched completely to the Si to create the reference regions with no resonance and a high level of reflection. In this case, once the dark current has been subtracted, the average value of the reflected light should be determined in each frame by averaging the level recorded from the reference regions and dividing the level from regions under test by it. The reference regions can be of various shapes, number, and position (see Figures 1-3 for examples). For example, the reference region can be disposed near an array of measurement regions (as in Figure 1) or within an array of measurement regions (as in Figures 2-3).

The following equation can be used as an example of how one might use the reference regions for normalizing data measured from an arrayed spot (or data spot). R_d is the light reflected from a pixel corresponding to an arrayed spot (in a measurement region), R_r is the light reflected from a pixel in the reference region, and d_pixel is the dark current on the corresponding pixels. Then, R_d' is the compensated intensity for the corresponding pixel in the arrayed spot: R_d' = ^fid"dp"^e' (Eq. 1)

Note that instead of one pixel, R_d and R₁. may represent the average or sum of many pixels in the arrayed spot and many pixels in the reference region. For example, one may choose to use the average of a substantially equal number of spots in the reference region as the arrayed spot to keep the noise contributions statistically similar.

R_d may also represent a pixel, an average of pixels, or a sum of pixels in the region neighboring the arrayed spot used as a reference for binding. Note that the neighboring area is a different type of reference region. The neighboring area PATENT

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is a reference to the binding that is occurring. The reference regions discussed herein are for source intensity normalization. The measurement of the binding reference areas will benefit from normalizing the intensity fluctuations from the source as well. If there are unwanted lateral interference fringes in the image, care should be taken to average over regions similar to the regions to be normalize, such that they are similarly affected by the lateral interference.

Example 1: SRIB

Slide preparation; To implement the present invention, the slide should be modified to create reference regions of a sufficiently different resonance condition. An illustration implementation consists of etching reference regions, such that the SiO₂ is totally removed and leaves only Si. This is particularly convenient since selective etching of these materials has been developed in the semiconductor industry, and standard protocols exist. Briefly, a mask for photolithography can be created as shown (Figure 4) to define the reference regions. A positive photoresist may be used, one selected for, along with a protocol for deep etching. Light exposure through the mask and subsequent developing of the resist and any necessary processing steps dictated by existing protocols provide a sample with resist everywhere except the reference regions. Etching with hydrofluoric acid (or using industry buffered oxide etch or 'BOE' for better performance) removes the SiO₂ in the reference regions. Beware that only appropriately trained personnel should handle HF or BOE given their extreme toxicity. Note that for wet etching using HF or BOE, the etchant will remove material laterally as well and hence sacrifice some region that is neither etched completely (like the reference region) nor non-etched such as the rest of the slide. However, the level of lateral etching, and the region lost to it, is generally an acceptable loss. In the present case with a 15 micron oxide, not much more than 15 microns around the region will be lost, whereas data spots (or array spots) PATENT

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are typically 150 microns or more in diameter and often placed 100 microns from adjacent features. The etch should be sufficient to remove all the SiO₂ ύi the photolithography defined regions. This is easy to accomplish, since HF or BOE selectively etches SiO₂ with a dramatically slower etch rate than for the Si layer below. One could also use deep reactive ion etching with standard protocols to provide less lateral etch.

Once the etching is complete, the sample must be washed thoroughly in water, and then thoroughly stripped of the photoresist. Care must be taken to ensure that the resist is sufficiently stripped. Steps include sonicatioii in acetone, rinse in acetone, ashing with Q-? plasma, and a light etch step briefly (few minutes) in 10% NaOH before being rinsed thoroughly in water. The importance of thoroughly stripping the resist is to ensure that subsequent capture agent immobilization chemistry is unaffected by the resist step.

Lastly, the slide should be prepared for a microarray experiment using immobilization chemistry, such as, e.g., epoxysilane, and then functionalized with arrayed capture agents, such as e.g., antibodies, protein, or DNA, to detect an analyte, such as e.g., a protein, a biomarker, RNA, DNA, mRNA, an antigen, or drug. Figure 5 shows a side view of a sample for SRTB, which includes a planar substrate with reference regions and measurement regions having capture agents. Apparatus: SRIB works by imaging reflected light from a structured sample

(Figure 6). The wavelength illumination light is stepped over time either by using a tunable laser or with a switchable light source, such that the light is substantially one wavelength and that that wavelength is switched between two, three, or more wavelengths. The substrate consists of Si with a 15 micron thick SiO₂ top surface thermally grown. The thickness of the SiO₂ layer may vary within any useful range. Exemplary ranges include from about 0.1 microns to about 50 microns, about 0.5 microns to about 25 microns, about 0.5 microns to about 50 microns, and about 5 microns to about 50 microns. PATENT

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An example apparatus and corresponding details have been reported in the literature (Ozkumur et al., supra). One embodiment is that if a reference photodetector is included for open loop post-processing compensation, it should be removed, or otherwise the signal from the detector should be disregarded in order to implement the present invention which will compensate by way of the reference regions. Note that if there is closed loop feedback on the source, it may remain in the system. Its use with the present invention would only serve to improve performance further. A second modification is that the light level and camera exposure should be appropriately set to avoid saturation of the camera at all the pixels, including the region under test and the reference regions for all wavelengths that will be used in the test. As is generally the case, a dark frame should be taken to determine the dark current and signal that results on each pixel in the absence of light.

Experiment protocol: The experiment to incubate with target molecules and record frames at different wavelengths at different times should be carried out as usual and as described in Ozkumur et al., supra. For the present invention, one must make sure that the reference regions are visible in the field of view for each frame captured on the camera and that the data from the reference regions are recorded along with the data from the arrayed region. Processing: First, the dark frame should be subtracted from all frames if it has not been done automatically either in the camera hardware or acquisition software. Next, the average recorded signal should be determined in the reference region. Optionally, the reference region closest to the region under test or an average of all the reference pixels in the frame can be determined. Care should be taken to exclude boarder regions of the reference region that may be only partially etched as described in the slide processing description. Each pixel in the data set (regions under test) for a given frame should be divided by the average intensity of the reference regions. The process normalizes the light level during the PATENT

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experiment as described herein. The normalized data values should then be processed as usual, which for SRIB, is fitting to the reflectivity model given in the literature in a least squares sense (Ozkumur et al, supra). Note that if the fitting algorithm employed expects signals of a particular magnitude, it may be necessary to multiply the resulting values by a single scalar factor to bring the normalized intensities into an expected range.

A possible additional step includes characterizing the wavelength dependency of the reference region and to compensate for this dependency during post-processing.

Example 2: RIfS

Described implementations of RIfS have imaged the reflected light from a thin film through a thick substrate (Gauglitz, supra). In this case, the present invention can be implemented by removing the top thin film with selective etching in defined reference regions (Figure 7). This is analogous to the example for SRIB, except that the materials are different. Note that there may be some wavelength dependency of the reflected light from the reference regions in this due to effects from the substrate. This can be characterized and accounted for in post-processing. A better option may be to divide the signals from the regions under test by the raw signal from the reference regions and adjust the model to eliminate the "IR" component discussed in the RIfS description which would now be normalized out as well (Gauglitz, supra).

One could also implement the present innovation for RItE by selectively etching the bottom substrate in regions. A dry etching technique could be employed in this case given the thickness of the substrate (more than 100 microns). As well, the reference regions may need to be larger so as to image the reference regions sufficiently without edge artifacts from the more than 100 micron tall vertical walls that would be created by etching down. PATENT

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Example 3: Picosope

In the described implementation of the Picoscope, there are no layers. The substrate is a solid thin SiO₂ slide, like a microscope coverslide (Nikitin et al., supra). In this case, a convenient implementation would be to etch reference regions so that they yield a substantially different interference pattern. Typical coverslide may be 200 microns thick. The slide can be patterned with photoresist and selectively etched in regions with HF or BOE as described in the SRIB example. Note that care must be taken to handle the slide delicately since it is fragile. For example, the etching could be stopped part way through when the slide has etched 100 microns into the 200 micron substrate. For an 850-nm source, the interference fringes from the region under test would be spaced about 0.9 nm apart in the spectrum. In the reference regions etched to 100 microns, the interference fringes would be spaced about 1.8 nm apart in the reflected spectrum. In the Picoscope system, there is an interference comb filter that is closely matched to the nominal substrate thickness and acts to pass or not pass many of the interference fringes. If the comb filter pass bands are also 0.9 nm spaced, then the 1.8 nm spaced fringes from the reference regions will encompass two pass bands and two stop bands per each node and anti-node of the reference region interference. As such, the reference regions will yield an average signal and be far less sensitive to changes in binding on the sample surface or slight adjustments of the interference filter. Thus, the reference regions, being insensitive to other changes, can serve as an accurate measure of the incident light intensity and compensate for intensity fluctuations.

Example 4: RCIB

In the described implementation of RCIB, there are two multilayer reflectors, one of which serves as the substrate for the DNA or protein array (Bergstein et al., supra). The present invention is for the inclusion of reference ATTORNEY DOCKET NO. 5071Θ/003WO2

regions for compensating source intensity fluctuations. To accomplish this, the local resonance condition should be substantially altered in a reference region. As an example, consider deep etching reference regions on the non-sample reflector (Figure 8). The reference regions could be defined by photolithography. The etching may need to be dry etched, such as reactive ion etching with the process parameters changes as one would etch through the different materials in the stack down to the Si substrate below. The system is then assembled with the non- sample reflector containing deeply etched regions. The resonance that would result at the deeply etched regions would exhibit considerably less cavity finesse since one reflector is simply Si, instead of a highly reflecting stack. The low finesse resonance would be slowly varying in wavelength compared to the sharp resonant curves from the high finesse region under test. As such, the intensity level of the light from the reference regions around the wavelength of the sharp finesse observed in nearby regions under test may be used to normalize the intensity of the incident light and compensate for source intensity fluctuations.

Example 5: SRIB and built up reference region

As a second example for SRIB, consider that instead of etching, one builds material up in the reference regions. One may add material by adding a bonded thin piece of glass such as pieces from a 200 micron thick coverglass. The interference pattern in the reflected light from these reference regions would have peaks in the spectra spaced by about 0.9 nm. One can illuminate with a source that is at least 0.9 nm broad or otherwise average steps over more than 0,9 nm for each measured point with a narrower source to effectively average over 0.9 nm. Averaging over 0.9 nm would give an average intensity that can be used to normalize source intensity fluctuations for the data from the region under test.

This concept of using a build up of material can be applied to other IOIM techniques. In the case of IUfS or picoscope, SiOa or a transparent polymer PATENT

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material (e.g., polydimethylsiloxane (PDMS)) could be added to the top surface in close proximity to a region under test. The added material would provide an increased thickness in that reference region and an interference pattern that could be averaged for a measure of the light intensity. The build up material can be any transparent material, as well as any material described herein. Exemplary materials include titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, and polymers (e.g., poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and plastic). Unlike the material for the region under test, a reference region with a material buildup need not be so flat and uniform. PDMS, PMMA, plastic, or other polymers, may be adhered with adhesive, or just pressed to the surface. For instance, PDMS can be bond the surface by using activated groups, but it can also just adhere by using vacuum or water bonding.

Example 6: Experimental setup for data collection

Figure 9 shows an exemplary setup for collecting data. The source consists of a tunable laser that steps on command from 765 nm to 782 nm in 1 nm increments with 0.01 nm accuracy and provides approximately 5 mW power at each step. The fiber optic cable is single mode fiber for 775 nm. The fused fiber Y coupler is designed for the wavelength ranged used and splits the light approximately even between the two outputs (50% / 50%). The photodetector is a silicon based photodiode that is biased and amplified to accurately record light intensities of ImW to 1OmW for the desired wavelength range. The fiber terminal is a simple FC-PC fiber coupler with the fiber only connected to one end. A 30mm focal length lens is positioned to provide at least 10 mm diameter of illumination on the sample. A spinning ground glass wheel diffuses the light further and removes spatial coherence (speckle) from the illumination that is still temporally coherent and substantially one wavelength (thin line width). The beam PATENT

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splitter is a pellicle beam splitter designed for the wavelength range that avoids ghosting and unwanted reflections. A beam dump serves to ensure that light is not reflected back into the system.

The sample is a Si (silicon) chip with approximately 17 microns of thermally grown oxide (silicon dioxide) and has been etched back in regions to form the reference regions (shown in Figures 1OA and 10B). The camera is a scientific digital camera that features low noise operation and efficient collection for the wavelength range used. The lens is a 60 mm focal length macro lens adjusted with additional spacing and focus to provide in focus 1 : 1 imaging of the sample surface. The wavelength is stepped in time, an image is taken at each wavelength recording the reflection from the sample, and images are transferred to the computer. For each pixel, the function of reflected intensity versus wavelength is considered and fit to a mathematical model to determine the oxide thickness on the sample (or sample phase delay) corresponding to that pixel. The result is a 2D map of thicknesses (or phase delay).

The mathematical model for data collection is also provided. The intensity versus wavelength function at a pixel fits the model given by Eq. 2:

where λ is the wavelength and R is the recorded intensity. B is the fitting parameter that is sought. A and C are additional fitting parameters that account for the curve amplitude and possible offset. r_}, r₂, and n are constants that depend on the refractive index of the materials and wavelength. The values of r_} and r₂ are related to the refractive index of the oxide (n), the buffer (n_b), and the silicon («_& ^■) by Eq. 3 and Eq. 4, PATENT

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,>?,, -!^• n Eq. 3

n ^■■ n i( r, = ^■ n + n Si Eq. 4

By using lookup iables, refractive indices according to wavelength are used for the values of n, n^, n^ such that the correct index is used for each input wavelength, λ. In the present case, n^. — 1.

Figures 10A and 1OB shows a sample chip that is 20 mm x 20 mm and contains regions under test that are 5 mm x 5 mm framed by an etched pattern that is 5.2 mm x 5.2 mm and 0.2 mm thick on two sides and 0.5 mm on two sides. The field of view is selected so as to encompass a region under test and at least some part of the surrounding etched frame and may vary from 0.05 mm to 100 mm in diameter to accomplish this criteria. The field of view illuminated in Figures 1 OA and 1OB is 10 mm in diameter and centered on a 5 mm x 5 mm region under test with the etched region also positioned within the field of view. The chip serves as the planar platform for the IOIM techniques described herein. Figure 1 OA is an actual CAD drawing used to design the chip. Figure 1OB is a schematic showing the different regions of the chip, where the areas in white are unetched and can be used for array spots. The areas in gray are etched and can be used as reference regions. Figure 1OB shows one exemplary embodiment of a reference region that is circumferentially disposed around a measurement region, where the area in gray surrounds the area in white. As indicated by the black box in Figure 1 IA, data do not have to be collected from the entire reference region but from a portion of the reference region (shown in black square). PATENT

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Example 7: Optical interference data normalized with data from reference regions

Data were obtained for a chip exposed to four different wavelengths. Figure 1 1 A provides a CCD image with a black box showing a portion of an etched region that will be used as the reference region. Figure 1 IB shows CCD images obtained at wavelengths 765 nm, 769 nm, 774 nm, and 779 nm. In Figures 1 IA and 1 IB, the center square is unetched and contains two faintly visible circle regions that are to be measured.

Figure 12 shows a model curve from Eq. 2, where n ~ 1.45 (corrected for dispersion at each wavelength), n_b = 1, n_Si ~ 1.37 (corrected for dispersion at each wavelength), A = 1, B = 17650, and C = 0.

Raw data were collected from three randomly selected pixels from within the measurement region of interest (Figure 13). The differences in amplitude and offset can be compensated by adjusting parameter A and C in the model. However, the shapes of the curves did not deviate significantly from the model curve. The shape of the curves should match the model for accurate fitting.

Voltage was recorded by the photodetector (PD) during the measurement for normalizing intensity noise (Figure 14). Figure 15 shows reflectivity data normalized by the PD voltage (intensity/voltage). Note that the curve shape has not improved, which indicates that source fluctuations are not the only source of noise.

Figure 16 shows the mean of reference region intensity as a function of wavelength. Since the oxide was etched to the Si layer, a nearly constant reflectivity is expected. The observed variation with wavelength indicates that some components not common to the photodetector are wavelength dependent. Components that could induce this wavelength dependence include the fiber splitter, lenses, the camera window, the camera sensitivity, and the beam splitter.

Figure 17 show the mean of reference region intensity versus PD voltage. The graph illustrates that a simple relationship between the two signals is not PATENT

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apparent and supports the argument that a wavelength dependence uncommon to these channels exists.

Figure 18 shows reflectivity data that has been normalized by the reference region mean (intensity /mean of reference region intensity). In contrast to the normalized data in Figure 15, the shape of the curves in Figure 18 closely resembled the model curve. The similarity in shape indicates that the noise source, namely the wavelength dependent component, has been compensated.

Figure 19 shows reflectivity data normalized by the mean of the reference region intensity and fit to the model. The curve shows a model curve from Eq. 2, where n ~ 1.45 (corrected for dispersion at each wavelength), n_b = 1 , n_Si ~ 1.37 (corrected for dispersion at each wavelength), A = 2.20, B = 17625.818, and C = 0.05.

The method and apparatus according to the invention provides an optical system that measures optical interference from a sample at multiple wavelengths and at multiple regions in parallel, and in which some (at least one) regions are designated as reference locations, whereby the interference has been appreciably altered for the purpose of compensating the measurement in the other (non- reference or measurement) regions against various common noise on the incident and/or reflected light intensity that affect all locations similarly. An optical system is described above where the interference signal is the result of thin Film interference and is monitored at numerous locations across a planar sample in parallel, and in which some (at least one) region has had the thin film thickness appreciably changed such that the interference curve generated is appreciably different from that being measured at the other regions and as such can be used to monitor and compensate common mode noise.

Further, an optical system is described above where the interference signal is the result of thin film interference and is monitored at numerous locations across a planar substrate in parallel, and in which some (at least one) region has had the PATENT

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thin film thickness etched away completely, such that there is no appreciable inference from those locations, only reflection from the substrate. As such, these reference regions can be used to monitor and compensate common mode noise. For all the disclosed methods, apparatuses, and optical systems, common noise sources include intensity fluctuations of the source, polarization effects, vibration and mechanical movement, and non-specific binding of additional material.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

What is claimed is:

Claims

PATENTATTORNEY DOCKET NO. 50710/003WO2

1. A planar substrate having a top face and a bottom face, said planar substrate comprising: a first specular reflecting interface at said top face of said planar substrate; a second specular reflecting interface that is substantially parallel to and underlies said first specular reflecting interface; one or more reference regions disposed on or within said first specular reflecting interface; and one or more measurement regions disposed on said first specular reflecting interface.

2. The planar substrate of claim 1 , further comprising: a first layer having a top face and a bottom face, wherein said first specular reflecting interface is at said top face of said first layer; and a second layer having a top face and a bottom face, wherein said top face of said second layer is connected to said bottom face of said first layer and said second specular reflecting interface lies between said first layer and said second layer.

3. The planar substrate of claim 1 , further comprising a first layer having a top face and a bottom face, wherein said first specular reflecting interface is at said top face of said first layer and said second specular reflecting interface is at said bottom face of said first layer.

4. The planar substrate of any of claims 2-3, wherein said first layer comprises one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide. PATENT

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5. The planar substrate of any of claims 2 or 4, wherein said second layer comprises one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.

6. The planar substrate of any of claims 1-5, wherein at least one reference region comprises a removed portion of said first specular reflecting interface.

7. The planar substrate of claim 6, wherein said removed portion exposes said second specular reflecting interface.

8. The planar substrate of any of claims 1-7, wherein at least one reference region comprises an added portion to said first specular interface.

9. The planar substrate of claim 8, wherein said added portion comprises one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(diraethylsiloxane), po3y(methyl methacrylate), or plastic.

10. The planar substrate of any of claims 1-9, wherein at least one reference region is disposed on or within said first specular reflecting interface at a distance of up to about 100 mm from at least one measurement region.

11. The planar substrate of any of claims 1-10, wherein at least one reference region is disposed circumferentially around at least one measurement region. PATENT

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12. The planar substrate of any of claims 1-10, wherein said measurement regions form an array and at least one reference region is disposed near or within said array.

13. The planar substrate of any of claims 1-12, wherein said planar substrate further comprises at least one capture agent directly or indirectly bound to said top face of said planar substrate.

14. A planar substrate for measuring optical interference, said planar substrate comprising: one or more first layers having a top face and a bottom face; one or more reference regions disposed on or within at least one first layer; and one or more measurement regions disposed on at least one first layer, wherein said one or more reference regions and said one or more measurement regions are disposed on or within at least one first layer to allow for simultaneously measuring said optical interference from said one or more reference regions and said one or more measurement regions.

15. The planar substrate of claim 14, wherein one or more first layers comprise one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide.

16. The planar substrate of claim 14, further comprising one or more second layers having a top face and a bottom face, wherein a top face of at least one second layer is connected to a bottom face of at least one first layer. PATENT

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17. The planar substrate of claim 16, wherein one or more second layers comprise one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.

18. The planar substrate of any of claims 14-17, wherein at least one reference region comprises a removed portion of at least one first layer to expose an underlying second layer or to decrease a height of said at least one first layer.

19. The planar substrate of any of claims 14-17, wherein at least one reference region comprises an added portion to at least one first layer.

20. The planar substrate of claim 19, wherein said added portion comprises one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic.

21. The planar substrate of any of claims 14-20, wherein at least one reference region is disposed on or within at least one first layer at a distance of up to about 100 mm from at least one measurement region.

22. The planar substrate of any of claims 14-21 , wherein at least one reference region is disposed circumferentially around at least one measurement region.

23. The planar substrate of any of claims 14-21 , wherein said measurement regions form an array and at least one reference region is disposed near or within said array. PATENT

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24. The planar substrate of any of claims 14-23, wherein said planar substrate further comprises one or more capture agents directly or indirectly bound to a top face of at least one first layer.

25. A planar substrate for measuring optical interference, said planar substrate comprising: a plurality of first layers, each layer having a top face and a bottom face; a plurality of second layers, each having a top face and a bottom face, wherein said plurality of first layers and said plurality of second layers form an alternating stack; and at least one reference region disposed within said alternating stack.

26. The planar substrate of claim 25, wherein said at least one reference region comprises a removed portion of at least one first layer to expose an underlying at least one second layer.

27. A method of determining an interference signal that is normalized to account for noise, said method comprising: a) providing a planar substrate comprising one or more reference regions and one or more measurement regions; b) reflecting a light source on said planar substrate; c) detecting an interference signal from said one or more reference regions and an interference signal from said one or more measurement regions; and d) adjusting said interference signal from said one or more measurement regions as a function of said interference signal from said one or more reference regions, wherein said adjusted interference signal is normalized to account for noise. PATENT

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28. The method of claim 27, wherein at least one reference region comprises a removed portion of said planar substrate.

29. The method of claim 28, wherein about 0.1 microns to about 50 microns of said planar substrate is removed.

30. The method of any of claims 27-29, wherein at least one reference region comprises an added portion to said planar substrate.

31. The method of claim 30, wherein said added portion comprises one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic.

32. The method of any of claims 27-31 , wherein said planar substrate comprises one or more first layers, each having a top face and a bottom face.

33. The method of claim 32, wherein said planar substrate further comprises one or more second layers, each having a top face and a bottom face, and wherein a bottom face of at least one first layer is connected to a top face of at least one second layer.

34. The method of claim 33, wherein said planar substrate comprises a plurality of first layers, each having a top face and a bottom face, and a plurality of second layers, each having a top face and a bottom face, and wherein said plurality of first layers and said plurality of second layers form an alternating stack. PATENT

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35. The method of any of claims 32-34, wherein one or more first layers comprise one or more oxides selected from the group consisting of silicon dioxide, titanium oxide, porous silicon, aluminum oxide, and tantalum pentoxide.

36. The method of any of claims 33-35, wherein one or more second layers comprise one or more metals selected from the group consisting of silicon, titanium, aluminum, tantalum, gold, silver, and tungsten.

37. The method of any of claims 32-36, wherein at least one reference region comprises a removed portion of at least one first layer to expose an underlying second layer or to decrease a height of said at least one first layer.

38. The method of claim 37, wherein about 0.1 microns to about 50 microns of said planar substrate is removed.

39. The method of any of claims 32-38, wherein at least one reference region comprises an added portion to at least one first layer.

40. The method of claim 39, wherein said added portion comprises one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic.

41. The method of any of claims 27-40, wherein said detecting step c) comprises detecting an interference signal from a portion of said one or more reference regions. PATENT

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42. The method of any of claims 27-41 , wherein said adjusting step d) comprises accounting for noise sources selected from the group consisting of intensity fluctuations of said light source, polarization effects, vibration, mechanical movement, and non-specific binding.

43. The method of any of claims 27-42, wherein said adjusting step d) comprises adjusting as a function of a mean of said interference signal from at least one reference region.

44. The method of any of claims 27-43, wherein said interference signal from at least one measurement region comprises a signal from a pixel, an average signal from more than one pixel, a sum signal from than one pixel, or a mean signal from more than one pixel.

45. The method of any of claims 27-44, wherein said interference signal from at least one reference region comprises a signal from a pixel, an average signal from more than one pixel, a sum signal from than one pixel, or a mean signal from more than one pixel.

46. A method of making a planar substrate comprising one or more reference regions and one or more measurement regions, said method comprising: a) masking a portion of said planar substrate to form a planar substrate having a mask, wherein said mask defines masked portions and exposed portions of said planar substrate; b) etching said planar substrate having said mask, wherein said exposed portions are etched to define at least one reference region and said masked portions define at least one measurement region; and PATENT

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c) removing said mask, wherein said at least one reference region and said at least one measurement region are disposed on said planar substrate to allow for simultaneously measuring optical interference from said at least one reference region and at least one measurement region.

47. The method of claim 46, wherein said masking step a) comprises applying photoresist to said planar substrate; applying a photomask to said photoresist and said planar substrate; exposing said photomask, said photoresist, and said planar substrate to a light source; and removing said photoresist that was not exposed to said light source to form said planar substrate having a mask.

48. The method of any of claims 46-47, wherein said etching step b) comprises hydrofluoric acid, buffered oxide etch, or deep reactive ion etching.

49. A method of making a planar substrate comprising one or more reference regions, said method comprising adding one or more materials on said planar substrate to form at least one reference region.

50. The method of claim 49, wherein said one or more materials are one or more of glass, silicon dioxide, titantium oxide, porous silicon, aluminum oxide, tantalum pentoxide, poly(dimethylsiloxane), poly(methyl methacrylate), or plastic.