Amplified multiple absorption spectroscopy

Abstract

An optically thin sample of sample material is analyzed by propagating probe electromagnetic radiation from a beam source along multiple different ray paths and directing each ray so that each ray path intersects on the sample at multiple different locations where the ray interacts with the sample material, causing modifications of the ray. The ray received at each of multiple detection volume regions is measured separately, and the measurements are analyzed to provide information about at least one property of the sample material at each interaction location. Analysis is performed to trace the path of the probe radiation from the position of the probe beam source to the detection volume region on the detection surface so as to identify the interaction locations and provide information about the presence of target material at each interaction location.

Description

[Technical Field]

[0001] The present invention relates generally to a multispectroscopic system for optically thin samples. The methods described herein can be used to measure the spectral properties of optically thin samples in general and biological samples in particular.

[0002] Related Applications This disclosure is related to U.S. Patent No. 8,345,254, Multiple Pass Imaging Spectroscopy, issued January 1, 2013 by the present inventors, hereinafter referred to as the MPIS patent.

[0003] This disclosure is related to U.S. Patent No. 10,585,044, High Efficiency Multiplexing, issued March 10, 2020 by the present inventors, hereinafter referred to as the HEMS patent.

[0004] This disclosure is related to U.S. Provisional Patent Application No. 63 / 059,298, entitled Multi-dimensional Spectroscopy, filed by the present inventors on July 31, 2020, hereinafter referred to as the MDS Patent, and currently filed on July 28, 2021 as PCT / CA2021 / 051057.

[0005] The disclosures of each of the above-cited patent applications may be consulted for further details. [Background technology]

[0006] The present invention relates to the spectral measurement of optically thin samples in general and biological samples in particular. Biological agents, such as bacteria and viruses, can be detected and identified at the genus, species, or strain level using spectroscopy. However, existing methods require relatively large sample sizes, on the order of micrograms, to provide spectra of sufficient quality for biological agent identification. Samples are typically prepared by culturing isolates to the required mass, which typically takes 24 to 72 hours. Conventional methods are further limited by their inability to handle samples in which several species are present. These drawbacks are partially overcome in the above-cited MPIS patent. The MPIS patent describes a multi-pass configuration that preserves spatial information through fixed amplification. In the MPIS configuration, a collimated beam of probe radiation is incident on multiple sample locations, and the amplitude of each beam is measured by a detector. The spatial information is recovered via a linear transformation. Optical amplification through multiple passes allows the measurement of the spectrum of a single bacterium or virus, eliminating the need for a culturing step. The spatial information allows the spectra of different species in the same sample to be separated and separately identified. The HEMS patent describes a multiplexed method for measuring spectral flux, where all of the spectral flux is modulated along multiple paths and the flux along each path is measured. The signal-to-noise ratio (SNR) is improved because all of the flux is measured and serial correlation between spectral bands is removed. Summary of the Invention

[0007] The invention may provide one or more of the following features.

[0008] A first objective of the present disclosure is to further improve the signal-to-noise ratio (SNR) of the above-described MPIS configuration by reducing the correlation between signals from adjacent sample locations.

[0009] A second object of the present disclosure is to provide an enhanced imaging method that includes magnification.

[0010] A third object of the present disclosure is to provide an amplification imaging method with variable amplification.

[0011] In accordance with the present invention, there is provided a method for analyzing an optically thin sample for the presence of a target material, comprising the steps of: disposing an optically thin sample potentially containing a target material; disposing a sample including a plurality of sample locations, each containing unknown sample material, known sample material, or no sample; propagating a beam of probe electromagnetic radiation from a beam source such that rays within the beam are directed along a plurality of different ray paths; directing each light beam using optical elements such that each light beam path intersects the sample multiple times at multiple different intersection locations; interacting the light beam with the sample material at each of a plurality of interaction locations to cause a modification of the light beam; directing each ray path to intersect a detection plane after intersecting the sample location, the detection plane being divided into a plurality of detection space regions; separately measuring the light beam received in each detection volume region, the measurements including information about the interaction of the sample material along the light beam path at each interaction location; analyzing the measurements to provide information regarding at least one property of the sample material at each interaction location; Including, A method is provided in which an analysis is performed to trace the path of probe radiation from a probe beam source position to the detection volume region on the detection surface so as to identify interaction locations and provide information regarding the presence of the target material at each interaction location.

[0012] The analysis therefore solves an inverse problem relating the amplitude measured at a point on the detector surface to the amplitude change at the identified interaction location.

[0013] The inverse problem can be solved by modeling the sample point amplitude as a linear combination of the detector point amplitude and the coefficients to be determined, where a first trial set of coefficients is generated by ray tracing simulation and iteratively varied to minimize the difference between the ray tracing simulation and the measured detector point amplitude. That is, this solution uses modeling of the detector point amplitude as a linear combination of the sample point amplitude variations. The term "ray" herein is a mathematical abstraction used in the field of geometric optics to describe a photon or packet of photons with a common Poynting vector. The present invention is described using geometric optics terminology to simplify the explanation of the key concepts of the present invention. The geometric optics approximation is valid for most practical cases where the size of the optical components is much larger than the wavelength of the electromagnetic radiation used. The geometric optics description is not limiting, as the formulation specified herein can be modified in a straightforward manner to use a physical optics description that takes into account diffraction effects.

[0014] The inverse problem can be solved by a neural network trained to model the relationship between sample area and detector output.

[0015] The inverse problem can be solved by defining a set of sample regions and a set of detector regions and binning together all rays that fall within each region resulting in a set of linear equations that can be solved to provide the inverse transform.

[0016] Preferably, at least one of the sample locations has a different shape than each of the detection locations.

[0017] Preferably, at least one of the sample positions is defined by at least one curve.

[0018] Preferably, at least one of the sample locations is defined by user selection.

[0019] In one embodiment, ray tracing is used to generate a sequence of different rays that traverse each sample position.

[0020] In one embodiment, the ray flux or number of traverses at the sample location can be dynamically adjusted according to the characteristics of the sample location.

[0021] In one embodiment, at least one beam of light traverses a pseudo-random sequence of sample positions.

[0022] In one embodiment, the multiple beams traverse different sequences of sample positions such that there is at least one different position in any two sequences.

[0023] In one embodiment, the optical layout is dynamically changed to provide different sequences of sample position intersections.

[0024] In one embodiment, the dynamic optical layout provides a spatial encoder.

[0025] In one embodiment, the at least one beam source further comprises a spatial modulator.

[0026] Preferably, the light beam is directed by the optics to intersect at least one of the sample locations at a plurality of different angles of incidence.

[0027] In one embodiment, at least one of the sample locations is discontinuous or comprises a plurality of different spots.

[0028] In one embodiment, the optical element is dynamically aligned so that the position of the laser beam reflected from the surface of the optical element on the detector corresponds to a desired position within the threshold.

[0029] In one embodiment, the number of times the beam crosses the sample position is varied to provide variable amplification.

[0030] In one embodiment, the optical element has a number of different configurations, each configuration of the optical element having a corresponding set of ray paths.

[0031] In one embodiment, the ray tracing is performed by a ray tracing model, where the probe beam is modeled as a set of rays that statistically reflect the properties of the probe radiation beam, each ray having an origin position on the source output port, each ray propagating through the optical system to an intersection with a detector surface, and each intersection with the sample material or optical element being stored.

[0032] In one embodiment, ray tracing is performed by modeling the flow of energy according to Maxwell's equations, with the direction of each ray at a location corresponding to the direction of the Poynting vector at that location.

[0033] In one embodiment, the sample material is disposed on a reflective surface, and multiple reflective surfaces are positioned above a plane of the sample material to reflect at least one beam of probe radiation that is incident on the sample material and interacts with the sample material multiple times.

[0034] In one embodiment, the sample material is disposed on a transparent substrate, and multiple reflective surfaces are disposed above and below the plane of the sample material to reflect at least one beam of probe radiation incident on the sample material and interacting multiple times with the sample material.

[0035] In one embodiment, at least a portion of the uncollimated beam of probe radiation is incident on the same sample region multiple times at the same mean angle of incidence interacting multiple times with at least one sample region, and the spatial distribution of the probe beam amplitude after said interactions is analyzed to provide information about the spatial distribution of interactions with the sample material.

[0036] In one embodiment, the set of sample regions included in at least one probe radiation path is varied based at least in part on the measured sample parameters.

[0037] Thus, optical configurations and methods are provided for multiplexing the interaction of electromagnetic radiation with an optically thin, inhomogeneous sample material. A beam of probe electromagnetic radiation is emitted by a radiation source, and light rays within the beam are guided by optical elements along multiple different ray paths, each ray path including m sample positions, where m is an integer greater than or equal to 2. The optical elements can have multiple different configurations in a reference coordinate system for the sample positions. That is, each configuration of the optical elements has a corresponding set of ray paths. The ray paths are selected so that the set of ray paths that intersect with the sample positions and terminate at measurement positions forms an invertible set of relationships between sample positions and measurement amplitudes. The different light rays can have different values ​​of m, as well as different wavelengths, phases, polarizations, and amplitudes. The sample positions may include unknown sample material, known sample material, or no sample at all. The ray paths may be arranged by the optical elements to impinge on the same sample position multiple times. At each sample position, the light rays interact with the material, causing at least one characteristic of the light rays (wavelength, amplitude, polarization, or phase) to change. After intersecting m sample locations, the ray path intersects a detection plane. The detection plane is divided into a plurality of spatial regions, and one or more properties (wavelength, amplitude, polarization, or phase) of the ray bundle intersecting each spatial region are measured by a measurement means. The measurements contain information about the interaction of the sample material along the ray path intersecting each measurement region. The measurements are analyzed to provide information about at least one property of the sample material at each sample location.

[0038] According to an important aspect of the present invention, a radiation source is provided. The radiation source provides an output beam of probing electromagnetic radiation having defined characteristics at one or more output ports. The output beam characteristics include a spectral distribution of wavelengths and a spatial distribution of phase, polarization, amplitude, and direction associated with each wavelength. The characteristics of each output beam of electromagnetic radiation can vary over time. The radiation source includes all optical elements necessary to generate the output beam with the defined characteristics. For example, the radiation source may include an interferometer that modulates the phase of each output wavelength. For example, the radiation source may include optical elements that spatially or temporally separate different wavelengths. For example, the radiation source may include a spatial modulator that varies the spatial distribution of the amplitude of the output beam. The radiation source may be, for example, a laser with beam-shaping optics. The radiation source may be, for example, a light-emitting device with beam-shaping optics. The radiation source may be, for example, a blackbody emitter with beam-shaping optics. The radiation source may include a filter that limits the wavelength range of the beam.

[0039] The present invention can be understood as a sequence of geometric transformations on a probe radiation beam by optical elements, which direct the probe radiation to interact with a sample material multiple times. The geometric transformations can be rotation, translation, inversion, reflection, scaling, or any combination thereof. The geometric transformations are mathematically inverted to provide information about the sample interaction. For example, the output beam of probe radiation can be a polychromatic beam with uniform intensity. The projection of the output beam onto the detection surface has uniform intensity and resembles a blank sheet of paper. The probe radiation beam is projected onto the sample material and interacts with the sample material to generate multiple images of the sample interaction. Each projection of the probe beam onto the sample material generates a different image. The probe beam image at the detector surface after multiple sample interactions is a superposition of all sample interactions. For example, if the sample interaction is absorption, the absorbance at each sample point is recorded at the corresponding point of the transformed probe beam.

[0040] The relationship between amplitude at sample material locations and detector surface locations can be determined using a ray tracing model. The rays are selected so that each ray (or group of rays) passes through a different set of sample locations and therefore carries information about a unique set of sample locations. Preferably, the sequence of sample locations for each ray (or group or rays) is a pseudo-random sequence of sample locations, as described in more detail below. Specifically, the probe beam is modeled as a set of rays that statistically reflect the properties of the probe radiation beam. Each ray has an origin location on the radiation source output port, a direction, a wavelength, a polarization, and a phase. The polarization vector carries the amplitude information. Each ray is propagated through the optical system to an intersection with the detector surface. Each intersection with the sample material or optical element is stored. The radiation propagation can be modeled based on the properties of ideal optical elements. Preferably, the properties used to model the optical elements are measured to account for the effects of impurities and geometric defects in the optical material (such as scratches, variations from an ideal surface, etc.). There are four approaches to solving the inverse problem, which is to relate the amplitude measured at a point on the detector surface to the amplitude at the sample surface point.

[0041] The first approach is to model the sample point amplitudes as a linear combination of the detector point amplitudes and coefficients to be determined. A first trial set of coefficients is generated and iteratively modified to minimize the difference between the ray trace simulation and the measured detector point amplitudes.

[0042] Second, a neural network can be trained to model the relationship between sample area and detector output.

[0043] A third approach is to define a set of sample regions and a set of detector regions and bin together all rays that fall within each region. This results in a set of linear equations that can be solved to provide the inverse transform.

[0044] A fourth approach is to generate an approximate solution using the second or third method, and then refine the solution so generated by the first method.

[0045] According to an important feature of the present invention, a plurality of redirecting elements operable to change the direction of probe radiation are provided. The redirecting elements may optionally change spatial characteristics of the probe radiation. The redirecting elements may be operable to direct probe radiation incident on a first sample location toward a second sample location, the first and second sample locations being successive sample locations in a sequence of sample locations on a path. The plurality of redirecting elements may be arranged in an array, with each successive redirecting element in the array directing radiation to the next sample location in the sequence of sample locations. The redirecting elements may be, for example, a single reflective surface. The redirecting elements may be, for example, a pair of reflective surfaces. The reflective surfaces may be internally reflective or externally reflective. The reflective surfaces may be planar or curved. The redirecting elements may be dynamically configured to direct received radiation from a first sample location to a second sample location for a first path and from the first sample location to a third sample location for a second path. For example, a redirecting element may direct radiation from sample position n to sample position n+1 for a first path and from sample position n to sample position n+3 for a second path. The redirecting element may be reconfigured, for example, by translating or rotating a mirror from a first position to a second position. The redirecting element may be reconfigured, for example, by electronically changing the optical properties of a surface from a reflective state to a transmissive state. The redirecting element may be reconfigured, for example, by electronically changing the refractive index of a material along the path to cause a change in refraction. The redirecting element may be reconfigured, for example, by changing the pitch of a diffractive element.

[0046] According to an important feature of the present invention, a set of sample positions is provided. Each sample position includes a device for positioning sample material at least during the measurement period. The sample positions may include a surface that externally reflects probe radiation. The surface may be flat or curved. Preferably, the reflectivity is greater than 90%. More preferably, the reflectivity is greater than 99%. The reflecting surface may be, for example, a gold mirror having a reflectivity greater than 99% for infrared probe radiation. The sample positions may include a surface that internally reflects probe radiation. Preferably, the internal reflection is total internal reflection, and evanescent waves from the probe radiation interact with sample material held close to the internal reflecting surface. The internal reflecting surface may be, for example, the internal surface of a crystal composed of Si, Ge, ZnSe, diamond, KRS-5, or other material from which an ATR device can be fabricated. The sample positions may include a surface that is transparent to the probe radiation. The probe radiation may be transmitted through both the sample material and the transparent surface. The transparent surface may be, for example, KBr, NaCl, CaF2, BaF2, AgCl, CsI, Si, Ge, ZnSe, or other optical window material. The transparent surface may be, for example, a thin polymer film or a thin glass plate. The transparent surface may be, for example, a gas stream.

[0047] According to an important feature of the present invention, a controller is provided that is operable to receive amplitude measurements for each path and analyze the measurements to provide information regarding a property of the sample material at at least one sample location. The property may be, for example, absorbance. The property may be, for example, scattering. The property may be, for example, luminescence. The property of the sample material may be calculated using measured amplitudes, theoretically calculated amplitudes (from known properties), or any combination thereof.

[0048] According to an important optional feature of the invention, which can be used independently of any of the above or below features, there is provided a translation device operable to generate relative motion between an array of sample locations and at least one redirecting element. For example, the array of redirecting elements can be displaced to discrete positions relative to the array of sample locations, each discrete position selecting a different path. The translation device can include a position measurement device. The translation device can be controlled in conjunction with a control device.

[0049] According to an important optional feature of the invention, which can be used independently of any of the above or below features, the controller may be further operable to select at least two different paths by configuring one or more optical elements, each path including a different sequence of sample positions. For example, the controller may generate a logic signal to cause a motor to rotate a mirror from a first angle to select a first path to a second angle to select a second path.

[0050] According to an important optional feature of the present invention, which can be used independently of any of the above or below features, there is provided an optical alignment device operable to measure at least one configuration parameter of a surface along a path. The optical alignment device may include a radiation source, a measurement device, and logic integral with a control device. For example, the optical alignment device may determine a normal to a mirror surface by directing a laser beam onto the surface and measuring the position of the reflected beam on a reference surface. The measured normal may be compared to a desired normal for a selected path, and the mirror configuration may be adjusted until the difference between the measured configuration and the desired configuration is less than a threshold value.

[0051] In an important embodiment that may be used in combination with any of the preceding or subsequent embodiments, the sample material is disposed on a reflective surface, and multiple reflective surfaces are positioned above the plane of the sample material to reflect at least one beam of probe radiation that is incident on the sample material and interacts with the sample material multiple times. The reflective surface may be, for example, a metallic first surface mirror. The reflective metallic surface may be, for example, Ag, Al, Au, Cu, Ni, or Pt.

[0052] In an important embodiment, which may be used in combination with any of the preceding or subsequent embodiments, the sample material is disposed on a transparent substrate, and a plurality of reflective surfaces are disposed above and below the plane of the sample material to reflect at least one beam of probe radiation that is incident on the sample material and interacts with the sample material multiple times. The transparent substrate may be, for example, glass, a crystal, a polymer film, or a gas stream.

[0053] In an important embodiment that may be used in combination with any of the preceding or subsequent embodiments, a sample material is disposed on a sample facet of an internal reflectivity element, the facet of the internal reflectivity element being arranged to direct at least one beam of probe radiation incident on the sample facet and interacting multiple times with the sample material proximate the sample facet. The beam of probe radiation may be mid-infrared radiation, for example, having a wavelength between 2.5 microns and 25 microns. The internal reflectivity element may be composed of a high refractive index material, such as, for example, Si, Ge, ZnSe, and ATMI-R5, CsI, or AgCl. Other materials may also be used.

[0054] In an important embodiment, which may be used in combination with any of the preceding or subsequent embodiments, at least a portion of the uncollimated beam of probe radiation is incident on the same sample region multiple times at the same mean angle of incidence interacting multiple times with at least one sample region, and the spatial distribution of the probe beam amplitude after said interactions is analyzed to provide information about the spatial distribution of interactions with the sample material.

[0055] In an important embodiment, which may be used in combination with any of the preceding or subsequent embodiments, at least a portion of the collimated beam of probe radiation is incident multiple times on the same sample region at different angles of incidence, and the spatial distribution of the probe beam amplitude after said interaction is analyzed to provide information about the spatial distribution of interactions with the sample material.

[0056] In an important embodiment, which may be used in combination with any of the preceding or subsequent embodiments, at least a portion of the uncollimated beam of probe radiation is incident multiple times on the same sample region at different angles of incidence, and the spatial distribution of the probe beam amplitude after said interaction is analyzed to provide information about the spatial distribution of interactions with the sample material.

[0057] In an important embodiment that may be used in combination with any of the preceding or subsequent embodiments, a sequence of M beams of probe radiation follow different paths that are incident on different subsets of sample regions selected from a set of N sample regions, the spatially distributed amplitude of each interacting probe radiation beam being analyzed to provide information about the spatial distribution of interactions with sample material within each sample region, each path in the sequence being incident on a different subset of less than N sample regions, at least one path in the sequence being incident on each sample region, and M is greater than or equal to N. Preferably, the set of sample regions included in each path is based on a cyclic permutation of a pseudo-random sequence. In some embodiments, at least one path in the sequence of paths is incident on the same sample region multiple times.

[0058] In important embodiments that may be used in combination with any of the preceding or subsequent embodiments, multiple probe radiation beams are incident simultaneously on a set of N sample regions, each probe radiation beam being incident on a different subset of less than N sample regions, each sample region being included in the path of at least one probe radiation beam. In some embodiments, at least one probe radiation beam is incident on the same sample region from different directions or at different angles of incidence. The optical configuration is changed at least N times, causing each probe radiation beam to interact with at least N different sequences of sample regions. A sequence of at least N measurements of spatial amplitude corresponding to the at least N different optical configurations is made for each probe radiation beam in the multiple simultaneous probe radiation beams after interaction with the sample material, and the spatial amplitudes are analyzed to provide information about the spatial distribution of sample interactions.

[0059] In an important embodiment that may be used in combination with any of the preceding or subsequent embodiments, the set of sample regions included in at least one probe radiation path is varied based at least in part on a measured sample parameter. For example, the spatial distribution of interactions with the sample material is approximated using a first set of probe radiation paths, and a second set of probe radiation paths is selected to improve the signal-to-noise ratio of the first approximation. For example, the second set of probe radiation paths may be selected to increase the total radiant flux in strongly absorbing sample regions, thereby improving photon counting statistics. For example, the second set of probe radiation paths may be selected to decrease the total radiant flux in weakly absorbing sample regions, thereby reducing the dynamic range required by the detection means and allowing greater amplification of weak signals.

[0060] In an important embodiment, which may be used in combination with any of the preceding or subsequent embodiments, the amplitude of probe radiation directed along at least one ray path is varied based at least in part on a measured sample parameter. For example, the amplitude measured after sample interaction along a first ray path may be significantly different from the amplitude measured after sample interaction along a second set of ray paths. The control means may generate a signal that causes a first set of ray path amplitude measurements to be made with low detector amplification and a wide dynamic range. The control means then selects measurement ray path amplitudes that differ from the average ray path amplitude by more than a threshold, and generates a signal that causes the probe radiation source to vary the initial amplitude of the probe radiation directed along the selected ray path so as to bring the amplitude of the probe radiation measured along the selected path closer to the average. The measured amplitudes along the selected paths may be scaled in subsequent numerical calculations to account for the change in initial amplitude. This rebalancing of the probe radiation amplitudes allows the amplification of the detector means to be adjusted to map the dynamic range of the detector to a smaller range of measurement amplitudes, thereby enabling the measurement of smaller amplitude differences between different ray paths. The probe radiation source may, for example, include an integrated spatial modulator that operates in time to direct or not direct radiation along a selected ray path. The percentage of time that the probe radiation is directed along a ray path determines the average initial amplitude of the probe radiation along that ray path. In another example, the spatial modulator may modulate the percentage of the probe radiation that is transmitted at a location in response to an electrical signal.

[0061] In an important embodiment, which may be used in combination with any of the preceding or following embodiments, the probe radiation beam is focused at least once during its interaction with the sample region.

[0062] In an important embodiment that may be used in combination with any of the preceding or subsequent embodiments, a path of probe radiation is traced from a probe beam source position to a position on the detection plane, and each sample position in the traced path is associated with the amplitude of the probe radiation at the detection plane. That is, the amplitude at a position on the detection plane is associated with the sum of interactions with the sample region included in the probe radiation path. This process is repeated for a representative sample along all paths from the probe radiation source to the detection plane. In some embodiments, the interaction with the sample material is modeled along each probe radiation path using test parameters, and the test parameters are iteratively varied to minimize the difference between the measured and calculated amplitudes at the detection plane. In some embodiments, the sample region is divided into multiple sample regions, and the detection plane is divided into multiple detection regions, and the sample regions are associated with the detection regions by a set of linear equations that are solved to provide information about the spatial distribution of interactions with the sample material. For example, the detection regions may be represented as rows of a matrix, and the sample regions may be represented as columns of the matrix. The amplitude (at the probe beam source) of each probe radiation path is summed in each column, corresponding to the sample region intersecting the probe radiation path in the row corresponding to the detection region. The resulting matrix equation can be solved by inversion, as described in the above-cited MPIS patent, or by least squares, as described in the above-cited HEMS patent. In some embodiments, the sample area is related to the detection surface area by training a neural network with calibration data, which is a set of known sample interactions at known sample areas.

[0063] In important embodiments that may be used in combination with any of the preceding or subsequent embodiments, the sample material is a biological material, which may be a eukaryotic cell, a prokaryotic cell, a virus particle, a bacterium, a tissue, a biopolymer solution, or any combination thereof.

[0064] In an important embodiment, which may be used in combination with any of the preceding or following embodiments, the sample material is a gas.

[0065] Implementation of the methods and systems of the present invention includes performing or completing selected tasks or steps manually, automatically, or a combination thereof. Furthermore, depending on the actual implementation and equipment of preferred embodiments of the methods and systems of the present invention, some selected steps may be implemented by hardware or software on any operating system, any firmware, or combination thereof. For example, as hardware, selected steps of the present invention may be implemented as a chip or circuit. As software, selected steps of the present invention may be implemented as multiple software instructions executed by a computer using any suitable operating system. In either case, selected steps of the methods and systems of the present invention may be described as being performed by a data processor, such as a computing platform for executing multiple instructions. [Brief explanation of the drawings]

[0066] [Figure 1A] FIG. 1A shows the prior art sampling geometry of the above-mentioned MPIS patent. [Figure 1B] FIG. 1B shows the cross section of the probe beam of FIG. 1A at the source. [Figure 1C] FIG. 1C shows a cross-sectional view of the sample area of ​​FIG. 1A. [Figure 1D] FIG. 1D is a schematic diagram of the configuration of FIG. 1A. [Figure 1E] FIG. 1E shows the cross section of the probe beam of FIG. 1A at the detector. [Figure 2A] FIG. 2A shows a sampling geometry according to the present invention. [Figure 2B] FIG. 2B shows the cross section of the probe beam of FIG. 2A at the source. [Figure 2C] FIG. 2C shows a cross-sectional view of the sample area of ​​FIG. 2A. [Figure 2D] FIG. 2D is a schematic diagram of the arrangement of FIG. 2A with a single probe beam. [Figure 2E] FIG. 2E shows the cross section of the probe beam of FIG. 2A at the detector. [Figure 3] FIG. 3 shows a schematic diagram of a sample area within a sample area of ​​an arrangement according to the invention. [Figure 4] FIG. 4 shows a schematic diagram of the configuration of the present invention. [Figure 5A] FIG. 5A shows a schematic diagram of the multiplexing configuration of the present invention. [Figure 5B] FIG. 5B shows a cross-sectional view of the configuration of FIG. 5A. [Figure 6A] FIG. 6A shows a first cross-sectional view of the beam path for multiple passes through the sample region. [Figure 6B] FIG. 6B shows a second cross-sectional view of the beam path for multiple passes through the sample region. [Figure 6C] FIG. 6C shows a third cross-sectional view of the beam path for multiple passes through the sample region. [Figure 7] FIG. 7 shows a plan view of an apparatus according to the invention for measuring spectra of a sequence of sample areas. DETAILED DESCRIPTION OF THE INVENTION

[0067] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. Detailed descriptions of structures or processes known in the art may be omitted to avoid obscuring the subject matter of the present disclosure. Furthermore, in the following description of the present disclosure, various specific definitions are provided to provide a general understanding of the present disclosure, and it is clear to those skilled in the art that the present disclosure can be implemented without such definitions.

[0068] FIG. 1A illustrates the sampling geometry of the above-cited MPIS patent, generally designated 10. Sample region 11 is at the center of unit sphere 12. A collimated beam of probe radiation 16 passes through a constant latitude line 13A and is incident on sample region 11 at a constant angle of incidence θ, designated 14. The projection of probe beam 16 onto the xy plane makes an angle phi with the x-axis, designated 15. Probe beam 16, as best seen in FIG. 1B, consists of a parallel (or nearly parallel) bundle of rays 21 distributed across beam cross-sectional area 16A. Ray bundle 21 is selected to correspond to a detector area. As shown in FIG. 1C, sample region 11 includes a region intersected by ray bundle 21 and having absorbing material 22. The probe radiation can be reflected or transmitted at sample region 11. As shown, probe radiation beam 16 is reflected as probe radiation beam 17. The cross-sectional areas of probe beam 16, shown at 16A, and probe beam 17, shown at 17A, are equal or nearly equal. On unit sphere 12, 16A and 17A subtend equal or nearly equal solid angles. On a first pass, probe beam 17 is redirected by a reflective optical element (not shown) as shown at 18 to form probe beam 19 toward sample region 11. Because the angle of incidence is constant, probe beam 19 lies on a constant line of latitude 13A. As shown, probe beam 19 transmits through sample region 11, and the transmitted beam, shown at 20, is incident on constant line of latitude 13B. Constant lines of latitude 13A and 13B are displaced by cos(theta) and -cos(theta), respectively, along the z-axis. The cross-sectional area of ​​probe beam 20, shown at 20A, is equal or nearly equal to the cross-sectional area of ​​probe beam 16, shown at 16A. In the second pass, the probe beam 17 is redirected by a constant translation vector to a second sample area 11B that is associated with the sample area 11. Each second successive sample area has a unit sphere 12 of the same type as described in association with the sample area 11.

[0069] As best seen in Figure 1D, the beam path can be parameterized along coordinate T. A radiation source 24 generates a collimated beam 16 that is initially incident on the sample region 11. As shown at 25, a geometric transformation on the beam is either reflected or transmitted by the sample region 11. In the case of reflection, the transformation may include beam segments 17 and 18, as shown in Figure 1A. The geometric transformation is performed by an optical element, which may be, for example, one or more mirrors or total internal reflection at one or more crystal planes. Note that the geometric transformation is relative to a fixed reference frame, such as the sample region. In the reference frame of the probe beam, the displacement between any two rays is invariant. Therefore, applying a sequence of geometric transformations to the sample region 11 produces the same result. Following geometric transformation 25, the probe beam 19 is incident on the sample region 11 twice. The sequence of receiving the interaction beam from the sample region, performing a geometric transformation on the probe beam, and incidenting the probe beam on the sample region is repeated multiple times. The Nth geometric transformation is shown at 26 which directs the probe beam 27 onto the sample region 11. The interacted beam is transformed by an optical element at 28 and is incident on a detector 29.

[0070] As best seen in Figure 1E, the probe beam at detector 29 is a superposition of the spatial pattern of absorption at sample location 11 (as shown in Figure 1C) and a sequence of geometric transformations. The pattern shown at 23 at detector 29 shows the effect of rotational motion only. In general, the pattern at detector 29 includes the effects of rotation, mirror reflection, inversion, and translation. In the MPIS patent cited above, each detector region is mapped to a set of sample subregions by a geometric transformation, and a matrix equation is solved to "unwind" the geometric transformation to obtain a sample plane image as shown in Figure 1C. The total absorption is multiplied by the number of sample passes N, thereby improving the signal-to-noise ratio for weakly absorbing sample materials. For reflectance, the probe beam must intersect either the line of constant latitude 13A or the line of constant latitude 13B, but not both, at least twice for each sample pass through sample region 11. Therefore, the maximum number of sample passes is π radians divided by the solid angle beamwidth. For transmission, the probe beam must intersect the constant latitude line 13A once and the constant latitude line 13B once for each sample pass through the sample region 11. Therefore, the maximum number of sample passes is 2*π radians divided by the solid angle beamwidth.

[0071] FIG. 2A shows the sampling geometry of the present invention, generally indicated at 30. Sample region 11 is at the center of unit sphere 12. Probe radiation can be incident on sample region 11 from any position on unit sphere 12; i.e., there are no restrictions on the spherical coordinate angles θ and phi, shown for beam 32 at 32T and 32P, respectively. In the MPIS patent mentioned above (FIG. 1A), theta is fixed and phi can be any angle. For comparison, the fixed angles of FIG. 1A are shown at 13A and 13B in FIG. 2A. Collimated beams, shown at 16, 17, 18, 19, and 20 (FIG. 1A), are within the scope of the present invention but are not replicated in FIG. 2A to avoid complexity.

[0072] As shown in FIG. 2A , probe radiation beams 31 and 35 are incident on sample region 11 at different angles of incidence 31T and 35T, respectively. Probe radiation beams 31 and 35 subtend solid angles 31A and 35A on unit sphere 12. In some embodiments suitable for general survey scans, solid angles 31A and 35A are equal. In some embodiments suitable for enhancing signal from selected subregions of sample region 11, solid angles 31A and 35A may be different. As shown in 31B, the probe radiation beams may be convergent, i.e., the cross-sectional area of ​​probe beam 31 at 31B is smaller than its cross-sectional area at 31A.

[0073] Probe beam 31 interacts with the material in sample region 11 and is transmitted as probe beam 32, subtending a solid angle 32A on unit sphere 12. Solid angles 31A and 32A are equal only in the special case of collimated light or converging light converging at the center point of the unit sphere. These special cases correspond to a magnification factor of 1. In general, solid angles 31A and 32A may be different. This important feature of the present invention allows the magnification of the sample region to be varied. Optical element 33, which may be, for example, a concave mirror, lens, or crystal facet, redirects and refocuses probe beam 32 as probe beam 34 toward second sample region 11C. In some embodiments, sample region 11C is related to sample region 11 by a non-constant translation vector, i.e., the displacement between the sample regions can vary. The spacing between the sample regions may be pseudo-random in a manner similar to the modulation regions discussed in the HEMS patent. That is, the probe beam is modulated by interacting with a pseudo-random sequence of sample regions. In other embodiments, the spacing between the sample regions is constant.

[0074] In an alternative configuration, probe beam 32 is directed by optical element 33 as probe beam 37 to optical element 45, which redirects probe beam 37 to sample region 11 as probe beam 39. Thus, probe beam segments 31, 32, 37, and 39 form a single probe beam that interacts twice with sample region 11. The pattern may be repeated multiple times to provide N sample passes. For each sample pass, the probe beam subtends an incident solid angle and an exit solid angle. In the case of transmission, the sum of the incident and exit solid angles is less than or equal to the solid angle of a unit sphere (4π steradians). This is in contrast to the MPIS case described above, where the sum of the incident and exit solid angles is twice the solid angle of a narrow ring at a given latitude. Because a larger solid angle is available, more sample passes are possible. In the case of reflection, the sum of the incident and exit solid angles is less than or equal to the solid angle of a hemisphere (2π steradians). This is in contrast to the MPIS case described above, where the sum of the entrance and exit solid angles is the solid angle of a narrow ring at a given latitude. Because a larger solid angle is available, more sample passes are possible.

[0075] FIG. 2B shows a cross-section of probe beam 35 at unit sphere surface 35A. The probe beam consists of rays with origin 42 displaced by displacement 44A from beam center 43. Each ray follows the Poynting vector for each point in the probe beam cross-section. In embodiments where the sample dimensions are larger than the wavelength of the probe radiation, the rays may be modeled by geometric optics methods. In some embodiments, the rays may be modeled by physical optics methods that include diffraction effects. In embodiments where the sample dimensions are similar to or smaller than the wavelength of the probe radiation, the rays may be modeled by solving Maxwell's equations. This may be done, for example, using a system of linear equations on an appropriately fine mesh.

[0076] FIG. 2C shows a cross-section of the probe beam 35 at its intersection with the sample region 11. The cross-sectional area is smaller due to the converging rays. As shown, ray 42 is displaced from the beam center 43 by a displacement 44B that is smaller than displacement 44A. The sample material 22 absorbs a portion of the incident energy flux. In a geometric model, rays intersecting the sample material 22 experience a constant absorbance. In a more rigorous physical model, the probability of absorption (absorption cross-section) of rays in close proximity to the sample material 22 varies with polarization and distance from the center of each absorbing molecule.

[0077] FIG. 2D shows a schematic diagram of a single-channel configuration of the present invention. Radiation source 24 generates a possibly converging probe radiation beam, as shown at 35, that is initially incident on sample region 11. Optical elements, shown generally at 38, redirect the transmitted probe beam 32 into segment 37, which is refocused to form probe beam 39. Probe beam 39 is incident on sample region 11 twice. The process of receiving the probe beam after interaction with the sample region, redirecting the beam, and optionally reshaping the beam, for example using a focusing mirror, is performed multiple times until final probe beam 41 is directed to be incident on the sample region for its Nth interaction. Interaction radiation is collected by optical elements 28 and directed to detector 29.

[0078] Figure 2E shows a schematic diagram of the radiation pattern on detector 29 after multiple interactions with the sample region. The illustrated detector 29 is scaled relative to sample region 11 in Figure 2C to illustrate the magnification features of this inventive configuration made possible by the focused probe beam. The radiation pattern 43 is analyzed to provide information about the distribution of absorbing material within sample region 11. The radiation amplitude at each point on the detector surface is related to the distribution of interacting material within the sample region by a sequence of geometric transformations that are non-commutative in all but a few special cases.

[0079] The detector surface is subdivided into a set of detector regions. For example, a detector region may correspond to a fixed pixel. For example, a detector region may correspond to a portion of the detection surface that is measured separately from other detector regions. The detector device may be configured, for example, as in the HEMS patents cited above.

[0080] FIG. 3 shows a schematic representation of the relationship between sample area, beam area, and sample region. Two sample regions are generally indicated at 50. Generally, there may be more than two sample regions, as shown in FIG. 4. Radiation source 24 generates radiation beam 47 having edge rays 45 and 46 and sample ray 70. Sample ray 70 intersects sample region 11 in sample region 54 at point 61 where ray 70 interacts with sample material located proximate point 61 (geometrical optics) or point 61 (physical optics). Ray 70 is reflected or transmitted as ray 71, and ray 72 is redirected by optical element 59 to be incident at point 62 where further interaction with the sample material may occur. Ray 72 is reflected or transmitted as ray 73, and ray 74 is redirected by optical element 59 to be incident at point 63 where further interaction with the sample material may occur. Ray 74 is reflected or transmitted as ray 75, and ray 76 is redirected by optical element 59 to be incident at point 64 where further interaction with the sample material may occur. Ray 76 is reflected or transmitted as ray 77, and ray 78 is redirected by optical element 59 to be incident at point 65 where further interaction with the sample material may occur. Ray 78 is reflected or transmitted as ray 79, and ray 80 is redirected by optical element 59 to be incident at point 66 where further interaction with the sample material may occur. Ray 80 is reflected or transmitted as ray 81 which is redirected by optical element 59 as ray 82 which is incident on detector 29. Ray 70R is therefore composed of ray segments 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, and 82. The symbol T collectively represents all segments of ray 70R within the scope of this disclosure. Light ray 70R interacts with the sample material at points 61, 62, 63, 64, 65, and 66; therefore, the total interaction is an average of six times the single-point interaction. Due to absorption, the absorbance is amplified by a factor of six in this example. In some embodiments, the amplification factor is greater than six. In the above-cited MPIS patents, the sample region is implicitly defined by the detector region. In the present invention, the sample interaction point can be assigned to any user-selected sample region. The selection of the sample region by the detector region is a special case in the present invention.

[0081] As shown in 11 and 11C, beam 47 intersects and interacts with multiple sample regions. Exemplary projections of beam 47 on sample region 11C, referred to as the beam region, are shown at 48 and 49. Generally, there can be more than two beam projections on the sample region. The beam projections include a common region 51 where sample material can interact with beam 47 twice. Generally, beam 47 can interact with sample material more than twice. In the MPIS patent (FIG. 1), there is a single beam that projects onto substantially all of the sample region. In the present invention, there can be multiple beams that individually project onto portions of the sample region but collectively project onto substantially all of the sample region. Generally, the beam interacts with a separate beam region within the sample region for each intersection of the beam and the sample region. At each intersection, the beam region can include all or a portion of the sample region.

[0082] Each sample area is subdivided into a set of user-defined sample areas. The sample areas may have a size defined, for example, by the spatial resolution of the optical system. In some embodiments, the sample area is subdivided into sample areas on a regular grid. This is shown in FIG. 3, where sample area 52 corresponds to the spatial resolution of the optical system and is arranged on a regular grid 53. As shown in 54, the sample area may be larger than the spatial resolution. Any sample area smaller than the sample area is acceptable. For example, a first sample area may occupy 99% of the sample area, and a second sample area may occupy 1% of the sample area. In this example, the first sample area may be the substrate material, and the second sample area may be the sample material. Sample area 54 is arranged on a regular grid 55A within sample area 11C, which logically extends to regular grid 55B within sample area 11. That is, a set of sample areas spans the sample area. In other embodiments, the sample area may be irregularly shaped, as shown by irregularly shaped sample area 56. Irregularly shaped sample regions can be defined by the shape of the sample material 22, as shown at 57. The sample region can also be discontinuous, consisting of two or more non-connected regions, as shown at 58A and 58B. A non-contiguous sample region, such as that shown at 58A and 58B, can correspond, for example, to a region occupied by a type of sample material having a spatial boundary that follows the contours of the sample material. The sample material can be, for example, a cell or a group of identical, non-connected cells. User-defined sample regions have three advantages. First, a user-defined sample region can interact with a larger radiant flux than a region defined by a regular grid, resulting in improved signal-to-noise. Second, the user-defined sample region boundaries can be selected to minimize spectral interference, thereby improving measurement specificity. Third, the number of user-selected sample regions can be fewer than the number of sample regions in a regular grid. This reduces the complexity of the inverse problem (relating detector amplitude to sample region interaction, as described in connection with Equation 1 below) and the computational resources required to solve the inverse problem.

[0083] The amplitude measured at each detector area of ​​detector 29 is a function of the material properties at each sample point intersected by the light ray propagating from the radiation source to that detector area. A i (λ)=ΣA k (λ)∫∫F(u j ,v j ,λ)G k (u j ,v j ,λ)du j domestic violence j (1) where A i (λ) is the amplitude at the ith detector region for wavelength λ, and A k (λ) is the amplitude of each ray at the source for each wavelength, and u j and v j is the spatial coordinate of the jth sample region. F(u j ,v j , λ) represents the sample absorbance as a function of space and wavelength, and G k (u j ,v j , λ) is the spatial distribution of the radiation amplitude associated with each ray of each wavelength. G k (u j ,v j , λ) can be measured directly in a calibration step, for example, by measuring the response from a sample material with known (preferably spatially invariant) properties, such as a mirror. k (u j ,v j , λ) can also be modeled at various sophistications, from geometrical optics ray tracing to physical optics to full Maxwell's equations models.

[0084] The ultimate goal of this invention is to j ,v j , λ). Three main approaches can be taken. First, Equation 1 can be approximated as a determinant, F(u j ,v j , λ) can be determined by solving a system of linear equations. Second, F(u j ,v j, λ) can be determined using a neural network algorithm. j ,v j , λ) can be determined using an iterative algorithm, and the A obtained for the model i (λ) of the sample material (and the corresponding F(u j ,v j ,λ) is modeled) and the approximate spatial distribution of F(u j ,v j ,λ) to iteratively adjust the model and A i The squared difference between the measured values ​​of (λ) is minimized. This second approach works best when the starting approximation is close to the final solution. The starting approximation can be provided, for example, by the solution of a matrix equation or a neural network algorithm.

[0085] G k (u j ,v j , λ) is approximately constant over the sample region or at least over a region of the sample region, Equation 1 can be expressed as the determinant d=Ms (2) where s is a vector of sample sub-region amplitudes, d is a vector of detector amplitudes, and M is a matrix of coefficients describing the contribution of each sample sub-region to the amplitude received at each detector sub-region. For example, for each ray that reaches a detector sub-region, the amplitude of that ray is added to the column representing each sample sub-region that the ray passed through. The matrix equation can be calculated by inverting (s=M -1 d) or can be solved by the least squares method.

[0086] FIG. 4 shows a schematic diagram of a configuration according to the present invention having multiple probe beams. A column shown at 91 contains m radiation sources, labeled B1 through Bm, where m is an integer greater than 1 and less than the number of sample regions, N. Each radiation source communicates with a controller 90, as shown at 92. Each radiation source is associated with a set of three-dimensional radiation paths, Ti, as shown at 93, where index i indicates the radiation source and bold text indicates that Ti is a matrix, with each radiation path in the radiation beam being a row and each column representing a coordinate along the radiation path. The number of interactions with a sample region, and therefore the number of coordinates included in each matrix, Ti, varies. As shown at 93, there are Ni sample region interactions, where i is the index of the radiation source. Ray paths may cross the same sample region multiple times from different directions. Each path contains a set of different sample regions, numbered 1 through Ni. Each sample region is included in at least one ray path. Different beam paths may include the same sample region, provided there is at least one sample region in the first path that is not included in the second path. The paths may each include a different number of sample regions. In the special case where the total sample region included in each path equals the number of sample regions, the configuration is similar to that in the above-cited HEMS patent, except that the amplitude at each sample region is not conserved but is modified by interaction with the sample material. In the more general case, the total sample region included in all beam paths is greater than the number of sample regions, N.

[0087] In principle, the number of ray paths in the beam approximates the number of photons, but in practice, a smaller number of ray paths may be used for numerical simulations. For example, the number of ray paths for numerical simulations may be in the range of 10,000 to 1,000,000. The controller 90 can operate on the beam source to modify the shape, amplitude, and spectral content of each beam. For example, the output beam may be shaped to compensate for the angle of incidence at the sample region. In this case, a cylindrical lens or mirror may shape the beam to an elliptical cross-section so that its projection on the sample region is circular. For example, the beam amplitude may be temporally modulated. Temporal modulation may be performed, for example, using an interferometer in which phase modulation is used to encode wavelengths. Temporal modulation may include a temporal sequence of wavelengths. In these cases, the temporal modulation is applied uniformly across the beam cross-section. The probe beam may be spatially modulated by a spatial modulator that is integral with the probe beam source or immediately following the probe beam source. In some embodiments, the spatial pattern is expanded by a focusing optics system by a factor less than one to generate a scaled, spatially modulated probe beam at the sample location. This feature can be used, for example, to selectively illuminate sample areas for calibration purposes.

[0088] As shown at 95, radiation source Bi directs radiation represented by a set of rays Ti that is incident on a first set of sample areas Si1. The radiation interacts with sample area Si1 and is collected by optical element Ei1, as shown at 96 in FIG. 2D and also shown at 38. Optical element Ei1 directs radiation that is incident on a second set of sample areas Si2, as shown at 97. The sequence of radiation incident on the sample areas and then on the optical elements is repeated a total of Ni times. The number of sample interactions N1, N2, and Nm for each of paths 1, 2, and m may be different. In some embodiments, the sample areas along each path are selected according to a pseudo-random sequence, as best shown in FIG. 7. In FIG. 7, a pseudo-random sequence {1,1,0,1,0,0,1} is shown, where the sample area at ordinal position "1" is included in the first ray path and the sample area at ordinal position "0" is included in the second ray path. In this example, N1 = 4 and N2 = 3. As shown at 98, the sample region S may optionally communicate with a controller 90 that controls external acoustic and electromagnetic fields in the sample region, as described in detail in the above-cited MDS patent. Briefly, the controller 90 sends a signal that imposes an external field on the sample region, causing periodic distortion of the sample molecular composition, and therefore spectral changes. In embodiments that include the MDS patent configurations in combination with features of the present disclosure, the MDS patent configurations increase the number of spectral features that can be measured, and the features of the present disclosure enhance the signal-to-noise ratio of those spectral features. The combined configuration may be used, for example, to measure the multidimensional spectrum of a virus, such as SARS-CoV2, where the present disclosure improves the sensitivity of the measurement and the configurations described in the MDS patents improve the specificity of virus identification.

[0089] As shown at 99, a controller 90 is optionally in communication with optical elements Eij and operable to change the configuration of the optical elements, where index i designates a radiation source and index j designates a set of sample regions at which the optical elements receive radiation. In some embodiments, the optical elements have a fixed configuration. For example, the optical elements may be surfaces of internally reflecting crystals. In some embodiments, the optical elements have a configuration selected by the controller 90 from a plurality of configurations. For example, a mirror may be moved from a first position to a second position, where the first position directs radiation to a first set of sample regions and the second position directs radiation to a second set of sample regions. In some embodiments, the optical elements have a fixed configuration and are translated or rotated from the first position to the second position relative to the sample region or set of sample regions. A first set of sample regions is selected at the first position and a second set of sample regions is selected at the second position. The optical elements may be any combination of mirrors, lenses, gratings, or others that operate to change a property of the radiation, where the property is selected from direction, phase, polarization, or amplitude.

[0090] As shown at 89, there are m detectors numbered 1 through m in communication with a controller 90. In some embodiments, the detector is a physical array having multiple transducers that convert radiation amplitude into an electrical signal. In some embodiments, the detector is a virtual array in which a single detector moves to multiple positions. In some embodiments, the detector is a single transducer.

[0091] FIG. 5A shows a schematic diagram of a multiplexing configuration of the present invention, generally designated 110. This configuration includes eleven sample regions equally spaced along row 115 in the x-direction given by coordinate frame 120. The sample regions are designated 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, and 131. As generally designated 140 in FIG. 5B, two layers of mirrors are arranged symmetrically about the z-axis, designated 136, on either side of the line of sample 115. The first layer of mirrors, designated 114 and 116 in FIG. 5A, have two configurations. In the first configuration, designated 114T, mirror 114 is rotated out of the path of beam 134 by an actuator (not shown) in communication with control unit 90, allowing the radiation beam to pass from sample line 115 to mirror 113 or vice versa. Similarly, mirror 116 can be rotated to configuration 116T by an actuator (not shown) in communication with control unit 90 to allow radiation beam 135 to pass from sample line 115 to mirror 117, or vice versa. A first configuration is shown by the dashed lines in Figure 5A, as indicated at 118. In a second configuration, shown at 114R and 116R in Figure 5B, mirrors 114 and 116 are rotated to reflect incident radiation towards sample line 115. Mirrors 113, 114R, 116R and 117 are oriented at the same angle relative to the z-axis.

[0092] As shown in FIG. 5A, a first incident radiation beam 111 is incident on sample area 121, reflected by a mirror of type 116R, and returned to the next sample position 122. The angle of incidence of radiation beam 111 is selected so that reflection from a mirror along line 114 or 116 advances one sample position, and reflection from a mirror along line 113 or 117 advances two sample positions. This scheme is easily extended to advance three or more sample positions per reflection, as shown at 137, by reflection between lines 113 and 114 or between lines 116 and 117. All that is required is that mirrors 114R and 116R are reflective on both sides. Thus, by this scheme, radiation beam 111 is incident successively on sample areas 121, 122, 124, 126, 127, and 130. The pattern on the sample area is a first code sequence 11010110010. Radiation beam 111 is measured by a detector at 132. Second radiation beam 112 is incident on sample areas 123, 125, 128, 129, and 131 and is measured by a detector at 133. The pattern on the sample area of ​​second beam 112 is the code sequence 00101001101. In the example shown in FIG. 5A, N1=6, N2=5, and N=11 for the scheme given in FIG. 4. By moving the mirrors along lines 114 and 116 between transmission and reflection positions, the device can be configured to generate cyclic permutations of the code sequence. The cyclic permutations of one code sequence and the associated amplitude measurements are easily recognized as the quasi-Hadamard case, and the absorbance of each sample area is obtained by matrix inversion of the code sequence. When beams 111 and 112 are measured, the absorbance of each sample area is obtained as described in detail in the HEMS patent cited above. Specifically, in the example given, there are 11 cyclic permutations of the mirrors, and each of the two detectors takes 11 measurements to generate a data vector y of length 22. The Z matrix described in the HEMS patent has 11 columns, one for each sample position, 11 rows (beams 111) for each cyclic permutation of the 22 pseudorandom sequence, and 11 rows (beams 112) for each complement of each cyclic permutation. From Equation 3 in the HEMS patent, the absorbance vector b is given by b = (Z TZ) -1 Z T y. The above works if the sample in each sample region is homogeneous. If it is heterogeneous, the sample region is divided into Q sample regions, radiation is traced from the source to the detector, and Equation 1 is solved for the absorbance in the sample region as before.

[0093] Figures 6A, 6B and 6C show cross-sections of beam paths with different angles of incidence on a sample area 142. The beam paths shown in Figures 6A, 6B and 6C are cross-sections through the yz plane as indicated by the coordinate system at 141. The incident radiation beam has a direction vector with a component in the x direction perpendicular to the plane of the figure.

[0094] In FIG. 6A , reflective surfaces are shown at 142, 143A, 143B, 144, 145A, 145B, and 146. Sample material is placed in sample region 142. Radiation travels sequentially between the reflective surfaces along paths 152 and 153, or portions thereof. Because an infinite number of paths along the x-axis are possible, only illustrative examples are shown. Note that the coordinate in the x-direction increases with each successive step in the given example. For example, radiation may be incident on sample region 142 and reflected to reflective surface 144 along path 152. At reflective surface 144, the radiation is reflected to reflective surface 143B and then to reflective surface 144. Radiation from reflective surface 144 is reflected back to sample region 142. At sample region 142, the radiation is reflected along path 153 and incident on reflective surface 145A. Reflecting surface 145A reflects the radiation onto sample area 142, where it is reflected by reflecting surface 143A. Reflecting surface 143A reflects the radiation onto sample area 142 where it is reflected along path 153 to reflecting surface 146. At reflecting surface 146, the radiation is reflected by reflecting surface 145B back to reflecting surface 146. The radiation is finally reflected from reflecting surface 146 back to sample area 142.

[0095] In Figure 6B, reflective surfaces are shown at 142, 147A, 147B, and 148. An infinite number of paths along the x-axis are possible, so only illustrative examples are shown. Radiation incident on sample area 142 is reflected off reflective surface 148 on path 154. Radiation is reflected from reflective surface 148 to reflective surface 147B and back to reflective surface 148, from which it is reflected back to sample area 142. Radiation reflected from sample area 142 is reflected off reflective surface 147A and back to sample area 142 from reflective surface 147A.

[0096] In Figure 6C, reflective surfaces are shown at 142, 149A, 149B, 150, 151A, 151B, and 152. As with Figure 6A, an infinite number of paths along the x-axis are possible, and the description of exemplary paths is similar to the example given in Figure 6A. In the description of Figure 6A, reflective surfaces 143A, 143B, 145A, 145B, 144, and 146 are replaced with reflective surfaces 149A, 149B, 151A, 151B, 150, and 152, respectively. Paths 152 and 153 are replaced with paths 155 and 156, respectively.

[0097] For each figure, the radiation beam is incident on the sample region 142 at a different displacement along the x-axis, as shown, for example, in FIG. 5A. The patterns of FIGS. 6A, 6B, and 6C may be combined so that the beam is incident on the same sample region multiple times at different angles of incidence. For example, the radiation beam may be incident on a sequence of sample regions along the x-axis according to the pattern of FIG. 6A. At the end of the sequence of FIG. 6A, the optical element reflects the radiation beam across the x-axis in the opposite direction according to the pattern of FIG. 6B, where it is incident on the same sequence of sample regions in reverse order. At the end of the sequence of FIG. 6B, the optical element reflects the radiation beam across the x-axis in the opposite direction according to the pattern of FIG. 6C, where it is incident on the same sequence of sample regions. In this illustrative example, the sample interaction at each sample region is increased by a factor of three. The concepts herein are easily extended by adding pairs of reflective elements at angles of incidence different from those shown.

[0098] FIG. 7 shows a plan view of a setup for measuring spectra of a sequence of sample regions, generally indicated at 160. The ordered sequence of sample regions 163 is carried on a movable platform 162, which moves from left to right in the figure as indicated at 164 over a reflective surface 161. In some embodiments (not shown), the movable platform 162 is integral with the reflective surface 161. Vertically displaced reflective surfaces are positioned on either side of the reflective surface 161, as shown in FIG. 5B or FIGS. 6A, 6B, and 6C. The sample region 163 contains sample material 163S. The sample material 163S may be, for example, a biological sample containing cells, bacteria, viruses, biological fluids, biopolymers, or any combination thereof. A first probe radiation beam 166 is emitted from a light source 165. The first probe beam 166 may optionally be modulated by a modulator 165M integral with the light source 165. Probe beam 166 is reflected in turn by reflective surfaces 167, 168, 169, 170, 171 and 172 before being received and analyzed by detector 173. A second probe radiation beam 175 is emitted from light source 174. Second probe beam 175 may optionally be modulated by modulator 174M integral with light source 174. Probe beam 175 is reflected in turn by reflective surfaces 176, 177, 178, 179 and 180 before being received and analyzed by detector 181. Each time radiation beam 166 crosses reflective surface 161, probe radiation beam 166 will either interact with and be reflected by a sample area if said sample area overlaps reflective surface 161, or will be reflected by reflective surface 161. Each time radiation beam 175 crosses reflective surface 161, probe radiation beam 175 either interacts with and is reflected by a sample area if said sample area overlaps reflective surface 161, or is reflected by reflective surface 161. In either case, the pattern of sample interaction and surface reflection forms a code sequence of 1s (sample interaction) and 0s (surface reflection). Reflective surfaces 167, 168, 169, 170, 171 and 172 are arranged such that translating movable platform 162 the length of one sample area provides a cyclic permutation of the first code sequence.Reflecting surfaces 176, 177, 178, 179, and 180 are arranged such that translating movable platform 162 the length of one sample region imparts a cyclic permutation of the second code sequence. The second encoding sequence is the complement of the first encoding sequence. In the illustrated example, the code sequence has a length of 7. Other, preferably longer, pseudorandom code sequences may be used. The minimum code sequence length in this configuration is 3.

[0099] The configuration of Figure 7 may be reconfigured by the controller 90 to change the code sequence. Each reflective surface may be reoriented as shown in detail for optical element 172. Optical element 172 has two reflective surfaces 172A and 172B and is rotated about axis 172X by an actuator (not shown) in communication with the controller 90. The angle between reflective surfaces 172A and 172B is fixed. A calibration beam source 182 in communication with the controller 90 generates a calibration beam 183 that is incident on reflective surface 172B. The reflected calibration beam 184 is measured by a position-sensitive detector 185 in communication with the controller 90. The angular displacement of reflective surface 172B is adjusted by the controller 90 until the angular displacement of calibration beam 184 corresponds to the angular displacement of reflective surface 172A required for the code configuration. The angular displacement of reflective surface 172A is related to the measured angular displacement of reflective surface 172B by a fixed angle. As shown, reflective surfaces 172A and 172B are parallel, but the method works with any known, fixed angle. In some embodiments, the reflective surfaces may be the same surface. Calibration beam source 182 may be, for example, a laser. The angular calibration of reflective surface 172A may be dynamically adjusted to compensate for misalignment caused, for example, by vibration. Alternatively, reflective surface 172A may be aligned to a desired configuration and then locked in place.

[0100] To measure the spectrum for each sample region, a spectrum is measured for each position of the moving platform within the code sequence. That is, a spectrum is measured at both detectors, and the platform is advanced by the length of one sample region. This process is repeated until the number of measurements at each detector equals the length of the code sequence. The spectrum thus measured is a combination of the spectra for each sample region. The spectrum for each sample region is obtained by the method described in the HEMS patent cited above. The same general method can be applied, with modifications, to the sample regions within the sample region. Generally, there is a group of sample regions from different sample regions that form a closed set along a ray path or a group of adjacent ray paths. The modification is that the sample region assigned to each detector region is selected by ray tracing.

[0101] Although the present configuration reduces the dynamic range of the measured light amplitude by summing absorption from different sample locations, some beam paths may contain only low-absorption sample regions. In this case, it may be advantageous to reduce the probe radiation amplitude along such low-absorption beam paths using a spatial modulator to limit the dynamic range of the amplitude at the detection means. Alternatively, it may be advantageous to increase the probe radiation amplitude along beam paths that intersect sample regions containing strongly absorbing sample material. The controller 90 may adjust the initial amplitude of the probe radiation along each beam path by sending appropriate control signals to modulators 165M and 174M.

[0102] The signal-to-noise ratio of each sample region spectrum depends in part on the bit resolution of the measured ray path amplitudes, or equivalently, the smallest measurable amplitude difference. In operation, the configuration of FIG. 7 may initially measure a sample region spectrum using a detector gain that includes all possible measured ray path amplitudes. The controller 90 analyzes the first set of measured ray path amplitudes and selects ray paths whose measured amplitudes differ from the average ray path amplitude by more than a threshold. The controller 90 generates and transmits signals to the modulators 165M and 174M, causing them to adjust the initial amplitude of the probe radiation along the selected ray paths such that the measured amplitude for the selected ray paths approaches the average amplitude measured for all ray paths. The controller 90 then maps the dynamic range of the detectors 173 and 181 to the (now smaller) expected range of ray path amplitudes. The controller 90 then causes a second set of measurements to be made, producing a second set of sample region spectra with an improved signal-to-noise ratio.

Claims

1. 1. A method for analyzing an optically thin sample for the presence of a target material, comprising: disposing the optically thin sample potentially containing the target material; The samples are each comprising a plurality of sample locations, each containing an unknown sample material, a known sample material, or no sample; propagating a non-collimated beam of probing electromagnetic radiation from a beam source such that rays within the beam are directed along a plurality of different ray paths; where each ray path is a parameterized path that includes multiple ray path segments in different directions; directing each light beam using optical elements such that each light beam path intersects the sample multiple times at multiple different intersection locations; interacting the light beam with the sample material at each of a plurality of interaction locations to cause a modification of the light beam; directing each ray path to intersect a detection plane after intersecting a sample location, the detection plane being divided into a plurality of detection volume regions; separately measuring the light beam received in each detection volume, the measurements including information about the interaction of the sample material along the light beam path at each interaction location; analyzing the measurements to provide information regarding at least one property of the sample material at each interaction location; Including, A method in which an analysis is performed to trace the path of a set of rays statistically representing the characteristics of the probe radiation beam from a position of a probe beam source to the detection space region on the detection surface to identify the interaction locations and provide information about the at least one property of the target material at each interaction location.

2. The method of claim 1 , wherein the analysis solves an inverse problem relating amplitudes measured at points on a detector surface to amplitudes at the identified interaction locations.

3. 3. The method of claim 2, wherein the inverse problem is solved by modeling sample point amplitudes as a linear combination of detector point amplitudes and coefficients to be determined, and a first trial set of coefficients is generated and iteratively varied to minimize the difference between ray tracing simulations and measured detector point amplitudes.

4. The method of claim 2 , wherein the inverse problem is solved by a neural network trained to model the relationship between sample area and detector output.

5. 3. The method of claim 2, wherein the inverse problem is solved by defining a set of sample regions and a set of detector regions and binning together all rays that fall within the respective regions resulting in a set of linear equations that can be solved to provide the inverse transform.

6. The method of any of claims 1 to 5, wherein the beam is converging or diverging to provide a magnification of the sample at the detection location.

7. The method according to any one of claims 1 to 6, wherein after at least one of said interactions, the beam is focused.

8. The method according to any one of claims 1 to 7, wherein at least one of the sample locations has a different shape than each of the detection locations.

9. The method of any preceding claim, wherein at least one of the sample locations is defined by at least one curve.

10. The method of any preceding claim, wherein at least one of the sample locations is defined by user selection.

11. The method according to any of the preceding claims, wherein the ray tracing is used to generate a sequence of different rays that traverse each sample position.

12. The method according to any of the preceding claims, wherein the beam flux or number of traverses at a sample location can be dynamically adjusted according to the characteristics of said sample location.

13. The method according to any one of claims 1 to 12, wherein at least one beam of light traverses a pseudo-random sequence of sample positions.

14. A method according to any preceding claim, wherein the multiple beams traverse different sequences of sample positions such that there is at least one different position in any two sequences.

15. The method of any preceding claim, wherein the optical layout is dynamically changed to provide different sequences of sample position intersections.

16. The method of claim 15 , wherein the dynamic optical layout provides a spatial encoder.

17. The method according to any one of claims 1 to 16, wherein at least one beam source further comprises a spatial modulator.

18. The method of any preceding claim, wherein the light beam is directed by an optical component to intersect at least one of the sample locations at a plurality of different angles of incidence.

19. The method of any preceding claim, wherein at least one of the sample locations is discontinuous.

20. The method of any preceding claim, wherein at least one of the sample locations comprises a plurality of different spots.

21. A method according to any preceding claim, wherein the optical element is dynamically aligned such that the position of the laser beam on the detector reflected from the surface of the optical element corresponds to a desired position within a threshold.

22. A method according to any preceding claim, wherein the number of times the beam crosses the sample position is varied to provide variable amplification.

23. The method of any preceding claim, wherein the optical element has a plurality of different configurations.

24. A method according to any preceding claim, wherein each arrangement of optical elements has a corresponding set of ray paths.

25. 25. A method according to any preceding claim, wherein the ray tracing is performed by a ray tracing model, wherein the probe beam is modelled as a set of rays that statistically reflect properties of the probe radiation beam, each ray having an origin position on a radiation source output port, each ray propagating through an optical system to an intersection with the detector surface, and each intersection with a sample material or optical element being stored.

26. 26. A method according to any preceding claim, wherein the ray tracing is performed by modelling the flow of energy by Maxwell's equations, the direction of each ray at a position corresponding to the direction of the Poynting vector at that position.

27. 27. A method according to any preceding claim, wherein the sample material is arranged on a reflective surface and a plurality of reflective surfaces are arranged above a plane of the sample material to reflect at least one beam of probe radiation incident on the sample material to interact with the sample material multiple times.

28. 28. A method according to any preceding claim, wherein the sample material is disposed on a transparent substrate, and wherein a plurality of reflective surfaces are arranged above and below a plane of the sample material to reflect at least one beam of probe radiation incident on the sample material to interact with the sample material multiple times.

29. 29. A method according to any preceding claim, wherein at least a portion of a non-collimated beam of probe radiation is incident on the same sample region multiple times at the same mean angle of incidence interacting multiple times with at least one sample region, and wherein the spatial distribution of the probe beam amplitude after said interactions is analysed to provide information about the spatial distribution of interactions with the sample material.

30. 30. The method of claim 29, wherein the sample region included in at least one probe radiation path is varied based at least in part on a measured sample parameter.

31. A method for analyzing an optically thin sample for the presence of a target material, comprising: disposing the optically thin sample potentially containing the target material; disposing the sample to include a plurality of sample locations, each of which contains unknown sample material, known sample material, or no sample; propagating a beam of probe electromagnetic radiation from a beam source such that rays within the beam are directed along a plurality of different ray paths; where each ray path is a parameterized path that includes multiple ray path segments in different directions; directing each light beam using optical elements such that each light beam path intersects multiple times on the sample at multiple different intersection locations; wherein the light beam is directed by an optical component to intersect at least one of the sample locations at a plurality of different angles of incidence; interacting the light beam with the sample material at each of a plurality of interaction locations to cause a modification of the light beam; directing each ray path to intersect a detection plane after intersecting a sample location, the detection plane being divided into a plurality of detection volume regions; separately measuring the light beam received in each detection volume, the measurements including information about the interaction of the sample material along the light beam path at each interaction location; analyzing the measurements to provide information regarding at least one property of the sample material at each interaction location; Including, A method in which an analysis is performed to trace the path of a set of rays statistically representing the characteristics of the probe radiation beam from a position of a probe beam source to the detection space region on the detection surface to identify the interaction locations and provide information about the at least one property of the target material at each interaction location.

32. A method for analyzing an optically thin sample for the presence of a target material, comprising: disposing the optically thin sample potentially containing the target material; disposing the sample to include a plurality of sample locations, each of which contains unknown sample material, known sample material, or no sample; propagating a beam of probe electromagnetic radiation from a beam source such that rays within the beam are directed along a plurality of different ray paths; where each ray path is a parameterized path that includes multiple ray path segments in different directions; directing each light beam using optical elements such that each light beam path intersects multiple times on the sample at multiple different intersection locations; wherein the rays within the beam converge or diverge to provide a magnification of the sample at the intersection location; interacting the light beam with the sample material at each of a plurality of interaction locations to cause a modification of the light beam; directing each ray path to intersect a detection plane after intersecting a sample location, the detection plane being divided into a plurality of detection volume regions; separately measuring the light beam received in each detection volume, the measurements including information about the interaction of the sample material along the light beam path at each interaction location; analyzing the measurements to provide information regarding at least one property of the sample material at each interaction location; Including, A method in which an analysis is performed to trace the path of a set of rays statistically representing the characteristics of the probe radiation beam from a position of a probe beam source to the detection space region on the detection surface to identify the interaction locations and provide information about the at least one property of the target material at each interaction location.

33. A method for analyzing an optically thin sample for the presence of a target material, comprising: disposing the optically thin sample potentially containing the target material; disposing the sample to include a plurality of sample locations, each of which contains unknown sample material, known sample material, or no sample; propagating a beam of probe electromagnetic radiation from a beam source such that rays within the beam are directed along a plurality of different ray paths; where each ray path is a parameterized path that includes multiple ray path segments in different directions; directing each light beam using optical elements such that each light beam path intersects multiple times on the sample at multiple different intersection locations; directing at least one beam of light across a pseudo-random sequence of sample positions; interacting the light beam with the sample material at each of a plurality of interaction locations to cause a modification of the light beam; directing each ray path to intersect a detection plane after intersecting a sample location, the detection plane being divided into a plurality of detection volume regions; separately measuring the light beam received in each detection volume, the measurements including information about the interaction of the sample material along the light beam path at each interaction location; analyzing the measurements to provide information regarding at least one property of the sample material at each interaction location; Including, A method in which an analysis is performed to trace the path of a set of rays statistically representing the characteristics of the probe radiation beam from a position of a probe beam source to the detection space region on the detection surface to identify the interaction locations and provide information about the at least one property of the target material at each interaction location.

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